Thrombosomes as an antiplatelet agent reversal agent

ABSTRACT

In some embodiments provided herein is a method of treating a coagulopathy in a subject, the method including administering to the subject in need thereof an effective amount of a composition including platelets or platelet derivatives and an incubating agent including one or more salts, a buffer, optionally a cryoprotectant, and optionally an organic solvent, wherein the subject has been treated or is being treated with an antiplatelet agent.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.16/994,377, filed Aug. 14, 2020 which claims priority to U.S.Provisional Application Ser. No. 62/887,923, filed on Aug. 16, 2019 andU.S. Provisional Application Ser. No. 63/065,337, filed on Aug. 13,2020, each of which is incorporated herein by reference in its entirety.

FIELD OF THE INVENTION

This disclosure serves to describe the use of thrombosomes as atreatment for drug-induced coagulopathy. The use of antiplatelet drugssuch as aspirin or clopidogrel can result in increased bleedingpotential. Here we demonstrate that thrombosomes can circumvent orovercome this inhibition to restore hemostasis.

BACKGROUND

Antiplatelet drugs (also herein called antiplatelet agents) are commonin the U.S. adult population and employ multiple mechanisms ofinhibiting platelet action. Antiplatelet drugs are used to treat and/orprevent a number of cerebrovascular and cardiovascular diseases.

Antiplatelet drugs, however, are responsible for many adversedrug-related events (ADEs). Overdose and adverse events related to thesedrugs carry the risk of serious bleeding and related complications inthe patient population. In addition, subjects treated with antiplateletdrugs face additional complications for surgery, as a subject may needto be tapered off the drugs before surgery, though cessation of therapycould put the subject at an increased risk for heart attack, stroke, ordeath.

There is therefore a need in the art for the treatment of coagulopathy,such as antiplatelet agent-induced coagulopathy, as well as a need for asolution for preparing subjects taking an anti-platelet drug forsurgery.

SUMMARY OF THE INVENTION

Provided herein in some embodiments is a method of treating acoagulopathy in a subject, the method including administering to thesubject in need thereof an effective amount of a composition includingplatelets or platelet derivatives and an incubating agent including oneor more salts, a buffer, optionally a cryoprotectant, and optionally anorganic solvent.

In some embodiments, provided herein is a method of treating acoagulopathy in a subject, the method including administering to thesubject in need thereof an effective amount of a composition prepared bya process including incubating platelets with an incubating agentincluding one or more salts, a buffer, optionally a cryoprotectant, andoptionally an organic solvent, to form the composition.

In some embodiments, provided herein is a method of restoring normalhemostasis in a subject, the method including administering to thesubject in need thereof an effective amount of a composition includingplatelets or platelet derivatives and an incubating agent including oneor more salts, a buffer, optionally a cryoprotectant, and optionally anorganic solvent.

In some embodiments, provided herein is a method of restoring normalhemostasis in a subject, the method including administering to thesubject in need thereof an effective amount of a composition prepared bya process including incubating platelets with an incubating agentincluding one or more salts, a buffer, optionally a cryoprotectant, andoptionally an organic solvent, to form the composition.

In some embodiments, provided herein is a method of preparing a subjectfor surgery, the method including administering to the subject in needthereof an effective amount of a composition including platelets orplatelet derivatives and an incubating agent including one or moresalts, a buffer, optionally a cryoprotectant, and optionally an organicsolvent. Implementations can include one or more of the followingfeatures. The surgery can be an emergency surgery. The surgery can be ascheduled surgery.

In some embodiments, provided herein is a method of preparing a subjectfor surgery, the method including administering to the subject in needthereof an effective amount of a composition prepared by a processincluding incubating platelets with an incubating agent including one ormore salts, a buffer, optionally a cryoprotectant, and optionally anorganic solvent, to form the composition. Implementations can includeone or more of the following features. The surgery can be an emergencysurgery. The surgery can be a scheduled surgery.

In some embodiments of the above methods, the subject has been treatedor is being treated with an antiplatelet agent. In some embodiments,treatment with the antiplatelet agent can be stopped. In someembodiments, treatment with the antiplatelet agent can be continued.

In some embodiments, provided herein is a method of ameliorating theeffects of an antiplatelet agent in a subject, the method includingadministering to the subject in need thereof an effective amount of acomposition including platelets or platelet derivatives and anincubating agent including one or more salts, a buffer, optionally acryoprotectant, and optionally an organic solvent.

In some embodiments, provided herein is a method of ameliorating theeffects of an antiplatelet agent in a subject, the method includingadministering to the subject in need thereof an effective amount of acomposition prepared by a process including incubating platelets with anincubating agent including one or more salts, a buffer, optionally acryoprotectant, and optionally an organic solvent, to form thecomposition.

In some embodiments, the effects of the antiplatelet agent can be theresult of an overdose of the antiplatelet agent.

In some embodiments, the antiplatelet agent can be selected from thegroup consisting of aspirin, cangrelor, ticagrelor, clopidogrel,prasugrel, eptifibatide, tirofiban, abciximab, and a supplement.

Some embodiments of any of the methods herein can include one or more ofthe following features. Administering can include administeringtopically. Administering can include administering parenterally.Administering can include administering intravenously. Administering caninclude administering intramuscularly. Administering can includeadministering intrathecally. Administering can include administeringsubcutaneously. Administering can include administeringintraperitoneally. The composition can be dried prior to theadministration step. The composition can be rehydrated following thedrying step. The composition can be freeze-dried prior to theadministration step. The composition can be rehydrated following thefreeze-drying step. The incubating agent can include one or more saltsselected from phosphate salts, sodium salts, potassium salts, calciumsalts, magnesium salts, and a combination of two or more thereof. Theincubating agent can include a carrier protein. The buffer can includeHEPES, sodium bicarbonate (NaHCO₃), or a combination thereof. Thecomposition can include one or more saccharides. The one or moresaccharides can include trehalose. The one or more saccharides caninclude polysucrose. The one or more saccharides can include dextrose.The composition can include an organic solvent. The platelets orplatelet derivatives can include thrombosomes.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows transmission light aggregometry of cangrelor (“Cang”) inplatelet rich plasma, expressed as the integrated aggregation curve,induced by 10 μM adenosine diphosphate (ADP) activation with and withoutincreasing concentrations of cangrelor.

FIG. 2 shows the effect of cangrelor, ADP, or a combination thereof onplatelet occlusion using T-TAS® technology.

FIG. 3 is a bar plot of the area under the curve (AUC) for data setsfrom FIG. 2 . Replicate data sets from FIG. 2 are presented as averages.

FIG. 4 is a bar plot of the occlusion time for data sets from FIG. 2 .Replicate data sets from FIG. 2 are presented as averages.

FIG. 5 shows the effect of thrombosomes (“thromb”; 300,000/μL)supplemented to platelet rich plasma in the presence and absence of ADPand cangrelor, at 60, 90, or 115 minutes post-rehydration on plateletocclusion using T-TAS® technology.

FIG. 6 is a bar plot of the AUC for data sets from FIG. 5 . Replicatedata sets from FIG. 5 are shown as averages.

FIG. 7 is a bar plot of the occlusion time for data sets from FIG. 5 .Replicate data sets from FIG. 5 are shown as averages.

FIG. 8 is a bar plot of the AUC from aggregation experiments forplatelets (at a concentration of 250,000 platelets per μL) treated withcollagen (10 μg/mL) and various concentrations of eptifibatide (“Epti”).

FIG. 9 shows the effect of eptifibatide at various concentrations onwhole blood using T-TAS® technology.

FIG. 10 shows the effect of thrombosome (“Tsomes”) supplementation(approximately 200,000/μL) on whole blood with and without variousconcentrations of eptifibatide using T-TAS® technology.

FIG. 11 is a bar plot of the occlusion time for the data sets from FIG.10 .

FIG. 12 is a bar plot of the AUC for the data sets from FIG. 10 .

FIG. 13 shows that thrombosomes (various lots) occlude in the presenceof eptifibatide in platelet-poor plasma (PPP).

FIG. 14 is a bar plot of the AUC for data sets from FIG. 13 . Replicatedata sets from FIG. 13 are shown as averages.

FIG. 15 is a bar plot of the occlusion time for the data sets from FIG.13 . Replicate data sets from FIG. 13 are shown as averages.

FIG. 16 is a bar plot of the AUC from aggregation experiments forplatelets treated with collagen (10 μg/mL) or arachidonic acid (“AA”;500 μg/mL) with and without various concentrations of aspirin (“ASA”).

FIG. 17 is a bar plot of the occlusion time for whole blood, whole bloodtreated with various concentrations of aspirin (ASA), and whole bloodtreated with various concentrations of aspirin and supplemented withthrombosomes (approximately 200,000-400,000/μL) as measured by responseto collagen coated plastic under shear using T-TAS® technology.

FIG. 18 shows the recovery of thrombus formation promoted bythrombosomes in whole blood in the presence of ASA (200 micromolar),cangrelor (1 micromolar), AP2 6F1 (40 micrograms), as measured byocclusion time on the T-TAS AR chip coated with thromboplastin andcollagen.

FIG. 19 shows the recovery of thrombus formation promoted bythrombosomes in whole blood in the presence of ASA (200 micromolar),cangrelor (1 micromolar) and 6F1 (40 micrograms/mL), as measured byocclusion (pressure) over time.

FIG. 20 shows the effect of thrombosomes supplementation toaspirin-(ASA-)inhibited whole blood (500 micromolar) on the interactionwith plastic immobilized porcine collagen under high shear, as measuredby AUC.

FIG. 21 shows the effect of thrombosomes supplementation toaspirin-(ASA-)inhibited whole blood (500 micromolar) on the interactionwith plastic immobilized porcine collage under high shear, as measuredby occlusion (pressure) over time.

FIG. 22 shows the effect of thrombosomes supplementation toaspirin-(ASA-)inhibited whole blood (100 micromolar) on the interactionwith plastic immobilized porcine collage under high shear, as measuredby AUC.

FIG. 23 shows the effect of thrombosomes supplementation toaspirin-(ASA-)inhibited whole blood (100 micromolar) on the interactionwith plastic immobilized porcine collage under high shear, as measuredby occlusion (pressure) over time.

FIG. 24 shows the effect on peak thrombin of thrombosome supplementationto normal and aspirin-inhibited plasma.

FIG. 25A shows the effect of cangrelor alone or cangrelor plusthrombosomes on platelet occlusion using T-TAS® technology.

FIG. 25B is a bar plot of the occlusion time for data sets from FIG.25A.

FIG. 26A shows the effect of tirofiban alone, or with random donorplatelets (RDP) or thrombosomes on platelet occlusion using T-TAS®technology.

FIG. 26B is a bar plot of the occlusion time for data sets from FIG.26A.

FIG. 27A shows the effect of eptifibatide alone, or with RDP orthrombosomes on platelet occlusion using T-TAS® technology.

FIG. 27B is a bar plot of the occlusion time for data sets from FIG.27A.

FIG. 28A shows the effect of AP2 alone, or with RDP or thrombosomes onplatelet occlusion using T-TAS® technology.

FIG. 28B is a bar plot of the occlusion time for data sets from FIG.28A.

FIG. 29A shows the effect of thrombosomes on PRP taken from a subject onaspirin therapy using T-TAS® technology.

FIG. 29B is a bar plot of the occlusion time for data sets from FIG.29A.

FIG. 30A shows the effect of thrombosomes on PRP taken from a subject onaspirin therapy on thrombin generation.

FIG. 30B is a bar plot of thrombin generation parameters for PRP takenfrom a subject on aspirin therapy, with or without added thrombosomes.

FIG. 31A shows aggregometry of PRP taken from a subject on ibuprofentherapy, with added buffer, arachidonic acid, or collagen.

FIG. 31B shows the effect of ADP on PRP taken from a subject onibuprofen therapy, with or without thrombosomes.

FIG. 32 shows the effect of dosing thrombosomes on the bleeding time ofmice treated with a superpharmacologic dose of clopidogrel.

DETAILED DESCRIPTION

Before embodiments of the present invention are described in detail, itis to be understood that the terminology used herein is for the purposeof describing particular embodiments only, and is not intended to belimiting. Unless defined otherwise, all technical and scientific termsused herein have the same meaning as commonly understood by one ofordinary skill in the art to which the term belongs. Although anymethods and materials similar or equivalent to those described hereincan be used in the practice of the present invention, the preferredmethods and materials are now described. All publications mentionedherein are incorporated herein by reference to disclose and describe themethods and/or materials in connection with which the publications arecited. The present disclosure is controlling to the extent it conflictswith any incorporated publication.

As used herein and in the appended claims, the singular forms “a”, “an”,and “the” include plural referents unless the context clearly dictatesotherwise. Thus, for example, reference to “a saccharide” includesreference to one or more saccharides, and equivalents thereof known tothose skilled in the art. Furthermore, the use of terms that can bedescribed using equivalent terms include the use of those equivalentterms. Thus, for example, the use of the term “subject” is to beunderstood to include the terms “patient”, “person”, “animal”, “human”,and other terms used in the art to indicate one who is subject to amedical treatment. The use of multiple terms to encompass a singleconcept is not to be construed as limiting the concept to only thoseterms used.

It is to be understood that the terminology used herein is for thepurpose of describing particular embodiments only, and is not intendedto be limiting. Further, where a range of values is disclosed, theskilled artisan will understand that all other specific values withinthe disclosed range are inherently disclosed by these values and theranges they represent without the need to disclose each specific valueor range herein. For example, a disclosed range of 1-10 includes 1-9,1-5, 2-10, 3.1-6, 1, 2, 3, 4, 5, and so forth. In addition, eachdisclosed range includes up to 5% lower for the lower value of the rangeand up to 5% higher for the higher value of the range. For example, adisclosed range of 4-10 includes 3.8-10.5. This concept is captured inthis document by the term “about”.

As used herein and in the appended claims, the term “platelet” caninclude whole platelets, fragmented platelets, platelet derivatives, orthrombosomes. “Platelets” within the above definition may include, forexample, platelets in whole blood, platelets in plasma, platelets inbuffer optionally supplemented with select plasma proteins, cold storedplatelets, dried platelets, cryopreserved platelets, thawedcryopreserved platelets, rehydrated dried platelets, rehydratedcryopreserved platelets, lyopreserved platelets, thawed lyopreservedplatelets, or rehydrated lyopreserved platelets. “Platelets” may be“platelets” of mammals, such as of humans, or such as of non-humanmammals.

As used herein, “thrombosomes” (sometimes also herein called “Tsomes” or“Ts”, particularly in the Examples and Figures) are platelet derivativesthat have been treated with an incubating agent (e.g., any of theincubating agents described herein) and lyopreserved (such asfreeze-dried). In some cases, thrombosomes can be prepared from pooledplatelets. Thrombosomes can have a shelf life of 2-3 years in dry format ambient temperature and can be rehydrated with sterile water withinminutes for immediate infusion. One example of thrombosomes areTHROMBOSOMES®, which are in clinical trials for the treatment of acutehemorrhage in thrombocytopenic patients. Typically, agents that inhibitFactor IIa, VIIa, IX, Xa, XI, Tissue Factor, or vitamin K-dependentsynthesis of clotting factors (e.g., Factor II, VII, IX, or X) or thatactivate antithrombin (e.g., antithrombin III) are considered to beanticoagulants. Other mechanisms of anticoagulants are known.Anticoagulants include dabigatran, argatroban, hirudin, rivaroxaban,apixaban, edoxaban, fondaparinux, warfarin, heparin, and low molecularweight heparins.

As used herein, an “antiplatelet agent” is an antithrombotic and doesnot include anticoagulants. Examples of antiplatelet agents includeaspirin (also called acetylsalicylic acid or ASA), cangrelor (e.g.,KENGREAL®), ticagrelor (e.g., BRILINTA®), clopidogrel (e.g., PLAVIX®),prasugrel (e.g., EFFIENT®), eptifibatide (e.g., INTEGRILIN®), tirofiban(e.g., AGGRASTAT®), and abciximab (e.g., REOPRO®). For the purpose ofthis disclosure, antiplatelet agents include agents that inhibit P2Yreceptors (e.g., P2Y₁₂), glycoprotein IIb/IIIa, or that antagonizethromboxane synthase or thromboxane receptors. Non-limiting examples ofthromboxane A₂ antagonists are aspirin, terutroban, and picotamide.Non-limiting examples of P2Y receptor antagonists include cangrelor,ticagrelor, elinogrel, clopidogrel, prasugrel, and ticlopidine.Non-limiting examples of glycoprotein IIb/IIIa include abciximab,eptifibatide, and tirofiban. NSAIDS (e.g., ibuprofen) are alsoconsidered to be antiplatelet agents for the purposes of thisdisclosure. Other mechanisms of anti-platelet agents are known.Antiplatelet agents also include PAR1 antagonists, PAR4 antagonists GPVIantagonists and alpha2beta1 collagen receptor antagonists. Non-limitingexamples of PAR-1 antagonists include vorapaxar and atopaxar. As usedherein, aspirin is considered to be an antiplatelet agent but not ananticoagulant. Additional non-limiting examples of antiplatelet agentsinclude cilostazol, prostaglandin E1, epoprostenol, dipyridamole,treprostinil sodium, and sarpogrelate.

In some embodiments, an antiplatelet agent can be selected from thegroup consisting of aspirin, cangrelor, ticagrelor, clopidogrel,prasugrel, eptifibatide, tirofiban, abciximab, and combinations thereof.In some embodiments, an antiplatelet agent can be selected from thegroup consisting of aspirin, cangrelor, ticagrelor, clopidogrel,prasugrel, eptifibatide, tirofiban, abciximab, terutroban, picotamide,elinogrel, ticlopidine, ibuprofen, vorapaxar, atopaxar, and combinationsthereof. In some embodiments, an antiplatelet agent can be selected fromthe group consisting of aspirin, cangrelor, ticagrelor, clopidogrel,prasugrel, eptifibatide, tirofiban, abciximab, terutroban, picotamide,elinogrel, ticlopidine, ibuprofen, vorapaxar, atopaxar, cilostazol,prostaglandin E1, epoprostenol, dipyridamole, treprostinil sodium,sarpogrelate and combinations thereof. In some embodiments, theantiplatelet agent can include multiple antiplatelet agents, such as 2(or more) of any of the antiplatelet agents described herein. In someembodiments, the antiplatelet agent can be aspirin and clopidogrel.

Cangrelor like clopidogrel, ticagrelor, and prasugrel, blocks the P2Y₁₂(ADP) receptor on platelets. Cangrelor can in some cases be used as arepresentative of this class of drug. Cangrelor, unlike clopidogrel andprasugrel, does not need hepatic metabolism to become biologicallyactive.

Eptifibatide is a peptide therapeutic that blocks the fibrin bindingrole of GPIIb-IIIa receptor on platelets. The drug is typicallyadministered via IV as a 180 μg/kg bolus followed by 2 μg/kg/mincontinuous infusion. The blood concentration of eptifibatide istypically about 1-2 μM. Bleeding times generally return to normal withinabout 1 hour of drug stoppage.

Aspirin is an irreversible cylcooxygenase (COX) inhibitor. The COXenzyme in platelets is responsible for synthesis of thromboxane A2,prostaglandin E2 and prostacyclin (PGI2). Aspirin permanentlyinactivates the COX enzyme within platelets, and since platelets do nothave the nuclear material to synthesize new enzyme, new platelets mustbe produced to overcome the aspirin effect. Without thromboxane A2,prostaglandin E2, and prostacyclin (PGI2) platelets are limited in theirpro-aggregation activity. Many people are maintained on a low dose ofaspirin to prevent unwanted clotting events. Aspirin bioavailabilitylargely varies with administration route, with a single 500 mg dose IVat peaks of 500 μM and the same dose orally at 44 μM.

The antiplatelet class of drugs is widely used to prevent unwantedclotting episodes that lead to heart failure, stroke, and the like. Inmany cases, an antiplatelet drug may need to be reversed or stopped. Inthe case of advanced notice, as in a pre-planned surgery situation, theantiplatelet drug dose can sometimes be stopped before the surgery,preventing unwanted bleeding during surgery. In the case where anantiplatelet agent needs reversing quickly, reversal agents aretypically not readily available, are expensive, or carry significantrisk to the patient. In the case of need for rapid antiplateletreversal, a platelet transfusion is typically administered, though theresponse to this is often only partial reversal. The caveat of thiscourse of reversal is that the newly-infused platelets themselves aresusceptible to circulating drug antiplatelet activity whereas, in someembodiments, compositions as described herein (e.g., includingthrombosomes) are not. In some embodiments, compositions as describedherein (e.g., including thrombosomes) are an active reversal agent. Insome embodiments, the hemostatic activity of compositions as describedherein (e.g., including thrombosomes) does not succumb to antiplateletdrugs.

Some exemplary antiplatelet agents and potential methods of reversal aredescribed below.

Acetylsalicylic acid (ASA; aspirin)—aspirin acts as a COX-1 blocker inplatelets, which renders the platelet inactive by irreversiblyinhibiting platelet-derived thromboxane formation. Clinically, aspirinis sometimes reversed by a platelet transfusion in emergency situationsor by stopping treatment where surgery is scheduled in the future.

Clopidogrel (e.g., PLAVIX®)—clopidogrel acts as to prevent ADP frombinding to its receptor on platelets. ADP binding leads to plateletshape change and aggregation. Clopidogrel is non-reversible. Clinically,clopidogrel is sometimes reversed by a platelet transfusion in emergencysituations or by stopping treatment where surgery is scheduled in thefuture.

Cangrelor (e.g., KENGREAL®)—cangrelor acts to prevent ADP from bindingto its receptor on platelets. ADP binding leads to platelet shape changeand aggregation. Clopidogrel is reversible and platelet function isreturned approximately 1 hour after stopping infusion. Clinically it isgenerally preferred when reversal is needed after a procedure.

Ticagrelor (e.g., BRILINTA®)—ticagrelor acts to prevent ADP from bindingto its receptor and acts as an inverse agonist. Ticagrelor is reversibleand platelet function can return after approximately 72 hours of thelast dosage. Reversal of action of ticagrelor can be affected by thetime after the last dose. If the last dose was longer than 24 hoursprevious, then platelet transfusion can sometimes be therapeutic toreverse the results.

Effient (e.g., PRASUGREL®)—Effient acts to prevent ADP from binding toits receptor and acts as a non-reversable antagonist. It being anon-reversible antagonist, new platelets must be formed to overcome itseffect. Clinically Effient is reversed by a platelet transfusion inemergency situations or by stopping treatment where surgery is scheduledin the future.

Eptifibatide (Integrilin)—Eptifibatide acts to block the GpIIb/IIIa andacts as a reversible antagonist. Clinically, Integrilin is reversed by aplatelet transfusion in emergency situations or by stopping treatmentwhere surgery is scheduled in the future.

Platelets infusions are currently used as a treatment method forantiplatelet drugs, but platelet transfusions only act to dilute out theeffect of these drugs. In some embodiments, thrombosomes are notreactive to these drugs and maintain their ability to aid in clotting.This makes treatment via thrombosomes entirely unique and introduces anew application for the product.

Platelet-derived products are not currently used as a treatment methodfor anticoagulant/antiplatelet drugs, and there are no currentlyapproved reversal agents for antiplatelet agents. As such, emergencytreatments (pre-op, trauma, and the like) are typically blanketprecautions to avoid or mitigate hemorrhage. Non-limiting examplesinclude infusion of plasma, red blood cells, and anti-fibrinolytics.Platelet derivatives (e.g., lyopreserved platelets (e.g., thrombosomes))may be an effective alternative or supplement to these generaltreatments.

Without being bound by any particular theory, it is believed thatthrombosomes can work at least in part by providing a procoagulantnegatively charged surface to augment thrombin generation above andbeyond that suppressed by the anti-coagulants. Similarly, without beingbound by any particular theory, it is believed that thrombosomes canwork at least in part by binding to and co-aggregating with circulatingplatelets.

Products and methods are described herein for controlling bleeding andimproving healing. The products and methods described herein can also beused to counteract the activity of an antiplatelet agent (e.g., aspirin(also called acetylsalicylic acid or ASA), cangrelor (e.g., KENGREAL®),ticagrelor (e.g., BRILINTA®), clopidogrel (e.g., PLAVIX®), prasugrel(e.g., EFFIENT®), eptifibatide (e.g., INTEGRILIN®), tirofiban (e.g.,AGGRASTAT®), or abciximab (e.g., REOPRO®)). The products and methodsdescribed herein are directed toward embodiments that can aid in theclosure and healing of wounds.

In certain embodiments, a composition comprising platelets such aslyophilized platelets or platelet derivatives may be delivered to awound on the surface of or in the interior of a patient. In variousembodiments, a composition comprising platelets such as lyophilizedplatelets or platelet derivatives can be applied in selected formsincluding, but not limited to, adhesive bandages, compression bandages,liquid solutions, aerosols, matrix compositions, and coated sutures orother medical closures. In embodiments, a composition comprisingplatelets such as lyophilized platelets or platelet derivatives may beadministered to all or only a portion of an affected area on the surfaceof a patient. In other embodiments, a composition comprising plateletssuch as lyophilized platelets or platelet derivatives may beadministered systemically, for example via the blood stream. Inembodiments, an application of the platelet derivative can producehemostatic effects for 2 or 3 days, preferably 5 to 10 days, or mostpreferably for up to 14 days.

Some embodiments provide a method of treating a coagulopathy in asubject, the method comprising administering to the subject in needthereof an effective amount of a composition comprising platelets suchas lyophilized platelets or platelet derivatives and an incubating agentcomprising one or more salts, a buffer, optionally a cryoprotectant(also called a lyophilizing agent), and optionally an organic solvent.

Some embodiments provide a method of treating a coagulopathy in asubject, the method comprising administering to the subject in needthereof an effective amount of a composition prepared by a processcomprising incubating platelets with an incubating agent comprising oneor more salts, a buffer, optionally a cryoprotectant, and optionally anorganic solvent, to form the composition.

In some embodiments of any of the methods described herein, thecoagulopathy is the result of an antiplatelet agent.

Some embodiments provide a method of treating coagulopathy in a subject,wherein the subject has been treated or is being treated with anantiplatelet agent, the method comprising administering to the subjectin need thereof an effective amount of a composition comprisingplatelets such as lyophilized platelets or platelet derivatives and anincubating agent comprising one or more salts, a buffer, optionally acryoprotectant, and optionally an organic solvent.

Some embodiments provide a method of treating coagulopathy in a subject,wherein the subject has been treated or is being treated with anantiplatelet agent, the method comprising administering to the subjectin need thereof an effective amount of a composition prepared by aprocess comprising incubating platelets with an incubating agentcomprising one or more salts, a buffer, optionally a cryoprotectant, andoptionally an organic solvent, to form the composition.

Some embodiments provide a method of restoring normal hemostasis in asubject, the method comprising administering to the subject in needthereof an effective amount of a composition comprising platelets suchas lyophilized platelets or platelet derivatives and an incubating agentcomprising one or more salts, a buffer, optionally a cryoprotectant, andoptionally an organic solvent.

Some embodiments provide a method of restoring normal hemostasis in asubject, the method comprising administering to the subject in needthereof an effective amount of a composition prepared by a processcomprising incubating platelets with an incubating agent comprising oneor more salts, a buffer, optionally a cryoprotectant, and optionally anorganic solvent, to form the composition.

Some embodiments provide a method of restoring normal hemostasis in asubject, wherein the subject has been treated or is being treated withan antiplatelet agent, the method comprising administering to thesubject in need thereof an effective amount of a composition comprisingplatelets such as lyophilized platelets or platelet derivatives and anincubating agent comprising one or more salts, a buffer, optionally acryoprotectant, and optionally an organic solvent.

Some embodiments provide a method of restoring normal hemostasis in asubject, wherein the subject has been treated or is being treated withan antiplatelet agent, the method comprising administering to thesubject in need thereof an effective amount of a composition prepared bya process comprising incubating platelets with an incubating agentcomprising one or more salts, a buffer, optionally a cryoprotectant, andoptionally an organic solvent, to form the composition.

Compositions as described herein can also be administered to prepare asubject for surgery, in some cases. For some patients taking anantiplatelet agent, it may be difficult or impossible to reduce thedosage of the antiplatelet agent before surgery (e.g., in the case oftrauma or other emergency surgery). For some patients taking anantiplatelet agent, it may be inadvisable to reduce the dosage of theantiplatelet agent before surgery (e.g., if the patient would be at riskof a thrombotic event (e.g., deep vein thrombosis, pulmonary embolism,or stroke) if the dosage of the antiplatelet agent were reduced overtime.

Accordingly, some embodiments provide a method of preparing a subjectfor surgery, the method comprising administering to the subject in needthereof an effective amount of a composition comprising platelets suchas lyophilized platelets or platelet derivatives and an incubating agentcomprising one or more salts, a buffer, optionally a cryoprotectant, andoptionally an organic solvent.

Some embodiments provide a method of preparing a subject for surgery,the method comprising administering to the subject in need thereof aneffective amount of a composition prepared by a process comprisingincubating platelets with an incubating agent comprising one or moresalts, a buffer, optionally a cryoprotectant, and optionally an organicsolvent, to form the composition.

Some embodiments provide a method of preparing a subject for surgery,wherein the subject has been treated or is being treated with anantiplatelet agent, the method comprising administering to the subjectin need thereof an effective amount of a composition comprisingplatelets such as lyophilized platelets or platelet derivatives and anincubating agent comprising one or more salts, a buffer, optionally acryoprotectant, and optionally an organic solvent.

Some embodiments provide a method of preparing a subject for surgery,wherein the subject has been treated or is being treated with anantiplatelet agent, the method comprising administering to the subjectin need thereof an effective amount of a composition prepared by aprocess comprising incubating platelets with an incubating agentcomprising one or more salts, a buffer, optionally a cryoprotectant, andoptionally an organic solvent, to form the composition.

In some embodiments, a surgery can be an emergency surgery (e.g., in thecase of trauma) or a scheduled surgery.

In some embodiments, treatment with an anticoagulant can be stopped(e.g., in preparation for surgery). In some embodiments, treatment withan anticoagulant can continue.

Some embodiments provide a method of ameliorating the effects of anantiplatelet agent in a subject, the method comprising administering tothe subject in need thereof an effective amount of a compositioncomprising platelets such as lyophilized platelets or plateletderivatives and an incubating agent comprising one or more salts, abuffer, optionally a cryoprotectant, and optionally an organic solvent.

Some embodiments provide a method of ameliorating the effects of anantiplatelet agent in a subject, the method comprising administering tothe subject in need thereof an effective amount of a compositionprepared by a process comprising incubating platelets with an incubatingagent comprising one or more salts, a buffer, optionally acryoprotectant, and optionally an organic solvent, to form thecomposition.

In some cases, the effects of an antiplatelet agent may need to beameliorated due to an incorrect dosage of an antiplatelet agent. Forexample, in some embodiments, the effects of an antiplatelet agent canbe ameliorated following an overdose of the antiplatelet agent. In somecases, the effects of an antiplatelet agent may need to be ameliorateddue to a potential for interaction with another drug (e.g., a secondantiplatelet agent). For example, in some embodiments, the effects of anantiplatelet agent can be ameliorated following an erroneous dosing oftwo or more drugs, at least one of which is an antiplatelet agent.

In some embodiments of any of the methods described herein, thecomposition can further comprise an active agent, such as ananti-fibrinolytic agent. Non-limiting examples of anti-fibrinolyticagents include ε-aminocaproic acid (EACA), tranexamic acid, aprotinin,aminomethylbenzoic acid, and fibrinogen. In some embodiments, plateletsor platelet derivatives can be loaded with an active agent, such as ananti-fibrinolytic agent.

Clotting parameters of blood (e.g., the subject's blood) can be assessedat any appropriate time during the methods described herein. Forexample, one or more clotting parameters of blood can be assessed beforeadministration of a composition comprising platelets such as lyophilizedplatelets or platelet derivatives as described herein, e.g., in order todetermine the need for administration of a composition comprisingplatelets or platelet derivatives as described herein. As anotherexample, one or more clotting parameters of blood can be assessed afteradministration of a composition comprising platelets or plateletderivatives as described herein, e.g., in order to determine theeffectiveness of the administered composition, to determine whetheradditional administration of the composition is warranted, or todetermine whether it is safe to perform a surgical procedure.

Accordingly, any of the methods described herein can include steps ofassessing one or more clotting parameters of blood before administrationof a composition comprising platelets or platelet derivatives asdescribed herein, assessing one or more clotting parameters of bloodafter administration of a composition comprising platelets or plateletderivatives as described herein, or both.

Any appropriate method can be used to assess clotting parameters ofblood. Non-limiting examples of methods include the prothrombin timeassay, international normalized ratio (INR), thrombin generation (TGA;which can be used to generate parameters such as, e.g., peak thrombin,endogenous thrombin potential (ETP), and lag time), thromboeleastography(TEG), activated clotting time (ACT), and partial thromboplastin time(PTT or aPTT).

Thrombin Generation

The thrombin generation assay measured the production of thrombin aftersample activation via a pro-coagulation agent resulting of thrombinenzymatic cleavage of a fluorescent peptide and release of fluorescentmolecule. The peak thrombin is a measure of the maximum thrombinproduced, lag time the time to start of thrombin production and ETP asthe total thrombin potentially produced.

In some embodiments, a patient can have a peak thrombin of about 60 nMto about 170 nM, such as about 65 nM to about 170 nM, such as about 65nM to about 120 nM, such as about 80 nM, before administration of acomposition comprising platelets or platelet derivatives as describedherein.

TEG assesses intrinsic hemostasis via plots of clot strength over time.Calcium chloride (CaCl₂)) is typically used as the initiating reagent. ATEG waveform (see, e.g., FIG. 16 ) has multiple parameters that canprovide information about clotting.

R-time=reaction time (s)—time of latency from start of test to initialfibrin formation.

K=kinetics (s)—speed of initial fibrin formation, time taken to achievea certain level of clot strength (e.g., an amplitude of 20 mm)

alpha angle=slope of line between R and K—measures the rate of clotformation.

MA=maximum amplitude (mm)—represents the ultimate strength of the fibrinclot.

A₃₀=amplitude 30 minutes after maximum amplitude is reached—representsrate of lysis phase.

In hypocoagulable blood states, R-time increases and MA decreases.R-time typically provides a broader response range than MA.

In the Total Thrombus-formation Analysis System (T-TAS®, FUJIMORI KOGYOCO., LTD), the sample is forced through collagen-coated microchannelsusing mineral oil. Changes in pressure are used to assess thrombusformation. The Occlusion Start Time is time it takes to reach 10 kPa,and the Occlusion Time=time it takes to each 480 kPa using an AR chip(e.g., Zacros Item No, TC0101). According to the manufacturer, an ARchip can be used for analyzing the formation of a mixed white thrombusconsisting chiefly of fibrin and activated platelets. It has a flow path(300 μm wide by 50 μm high) coated with collagen and tissue factors andcan be used to analyze the clotting function and platelet function. Incomparison, a PL chip can be used for analyzing the formation of aplatelet thrombus consisting chiefly of activated platelets. A PL chiphas a flow path coated with collagen only and can be used to analyze theplatelet function.

The ACT assay is the most basic, but possibly most reliable, way tomeasure clotting time (t_(ACT)), determined by a magnet's resistance togravity as a clot forms around it. Typical donor blood has at_(ACT)˜200-300 s using only CaCl₂).

Some embodiments provide a method of increasing thrombin generation in asubject, the method comprising administering to the subject in needthereof an effective amount of a composition comprising platelets suchas lyophilized platelets or platelet derivatives and an incubating agentcomprising one or more salts, a buffer, optionally a cryoprotectant, andoptionally an organic solvent.

Some embodiments, provide a method of increasing thrombin generation ina subject, the method comprising administering to the subject in needthereof an effective amount of a composition prepared by a processcomprising incubating platelets with an incubating agent comprising oneor more salts, a buffer, optionally a cryoprotectant, and optionally anorganic solvent, to form the composition.

Some embodiments provide a method of increasing peak thrombin in asubject, the method comprising administering to the subject in needthereof an effective amount of a composition comprising platelets suchas lyophilized platelets or platelet derivatives and an incubating agentcomprising one or more salts, a buffer, optionally a cryoprotectant, andoptionally an organic solvent.

Some embodiments provide a method of increasing peak thrombin in asubject, the method comprising administering to the subject in needthereof an effective amount of a composition prepared by a processcomprising incubating platelets with an incubating agent comprising oneor more salts, a buffer, optionally a cryoprotectant, and optionally anorganic solvent, to form the composition.

In some embodiments, prior to the administering, the peak thrombin ofthe subject was below 66 nM (e.g., below 64 nM, 62 nM, 60 nM, 55 nM, 50nM, 45 nM, 40 nM, 35 nM, 30 nM, 25 nM, 20 nM, 15 nM, 10 nM, or 5 nM). Insome embodiments, after the administering, the peak thrombin of thesubject is above 66 nM (e.g., above 68 nM, 70 nM, 75 nM, 80 nM, 85 nM,90 nM, 95 nM, 100 nM, 110 nM, 120 nM, 130 nM, 140 nM, or 150 nM). Insome embodiments, after the administering, the peak thrombin of thesubject is between 66 and 166 nM. Peak thrombin can be measured by anyappropriate method.

An “effective amount” as used herein is an amount of the compositionthat comprises an amount of platelets such as lyophilized platelets orplatelet derivatives (e.g., thrombosomes) effective in treating thesubject. Such an amount of platelets or platelet derivatives (e.g.,thrombosomes) includes any appropriate dosage of a compositioncomprising platelets or platelet derivatives as described herein thatcan be administered to the subject. For example, in some embodiments, adose of a composition comprising platelets or platelet derivatives(e.g., thrombosomes) can include about 1.0×10⁷ particles to about1.0×10¹⁰ particles, such as about 1.6×10⁷ particles (e.g.,thrombosomes)/kg to about 1.0×10¹⁰ particles/kg (e.g., about 1.6×10⁷ toabout 5.1×10⁹ particles/kg, about 1.6×10⁷ to about 3.0×10⁹ particles/kg,about 1.6×10⁷ to about 1.0×10⁹ particles/kg, about 1.6×10⁷ to about5.0×10⁸ particles/kg, about 1.6×10⁷ to about 1.0×10⁸ particles/kg, about1.6×10⁷ to about 5.0×10⁷ particles/kg, about 5.0×10⁷ to about 1.0×10⁸particles/kg, about 1.0×10⁸ to about 5.0×10⁸ particles/kg, about 5.0×10⁸to about 1.0×10⁹ particles/kg, about 1.0×10⁹ to about 5.0×10⁹particles/kg, or about 5.0×10⁹ to about 1.0×10¹⁰ particles/kg).

In some embodiments of the methods herein, the composition isadministered topically. In some embodiments, topical administration caninclude administration via a solution, cream, gel, suspension, putty,particulates, or powder. In some embodiments, topical administration caninclude administration via a bandage (e.g. an adhesive bandage or acompression bandage) or medical closure (e.g., sutures, staples)); forexample the platelet derivatives (e.g., lyopreserved platelets (e.g.,thrombosomes)) can be embedded therein or coated thereupon), asdescribed in PCT Publication No. WO2017/040238 (e.g., paragraphs[013]-[069]), corresponding to U.S. patent application Ser. No.15/776,255, the entirety of which is herein incorporated by reference.

In some embodiments of the methods herein, the composition isadministered parenterally.

In some embodiments of the methods herein, the composition isadministered intravenously.

In some embodiments of the methods herein, the composition isadministered intramuscularly.

In some embodiments of the methods herein, the composition isadministered intrathecally.

In some embodiments of the methods herein, the composition isadministered subcutaneously.

In some embodiments of the methods herein, the composition isadministered intraperitoneally.

In some embodiments of the methods herein, the composition is driedprior to the administration step. In some embodiments of the method, thecomposition is freeze-dried prior to the administration step. In someembodiments of the method, the composition is rehydrated following thedrying or freeze-drying step.

In some embodiments, the antiplatelet agent is selected from the groupconsisting of aspirin (also called acetylsalicylic acid or ASA); a P2Y12inhibitor such as cangrelor (e.g., KENGREAL®), ticagrelor (e.g.,BRILINTA®), clopidogrel (e.g., PLAVIX®), or prasugrel (e.g., EFFIENT®);a glycoprotein IIb/IIIa inhibitor such as eptifibatide (e.g.,INTEGRILIN®), tirofiban (e.g., AGGRASTAT®), or abciximab (e.g.,REOPRO®)); supplements such as herbal supplements; or a combination ofany thereof. Examples of supplements include ginger, ginseng, ginkgo,green tea, kava, saw palmetto, boldo (Peumus boldus), Danshen (Salviamiltiorrhiza), Dong quai (Angelica sinensis) papaya (Carica papaya),fish oil, and vitamin E. Examples of herbal supplements include ginger,ginseng, and ginkgo.

In some embodiments, the antiplatelet agent is aspirin.

In some embodiments, the antiplatelet agent is cangrelor (e.g.,KENGREAL®).

In some embodiments, the antiplatelet agent is ticagrelor (e.g.,BRILINTA®).

In some embodiments, the antiplatelet agent is clopidogrel (e.g.,PLAVIX®).

In some embodiments, the antiplatelet agent is prasugrel (e.g.,EFFIENT®).

In some embodiments, the antiplatelet agent is eptifibatide (e.g.,INTEGRILIN®).

In some embodiments, the antiplatelet agent is tirofiban (e.g.,AGGRASTAT®).

In some embodiments, the antiplatelet agent is abciximab (e.g.,REOPRO®).

In some embodiments, the antiplatelet agent is terutroban.

In some embodiments, the antiplatelet agent is picotamide.

In some embodiments, the antiplatelet agent is elinogrel.

In some embodiments, the antiplatelet agent is ticlopidine.

In some embodiments, the antiplatelet agent is ibuprofen.

In some embodiments, the antiplatelet agent is vorapaxar.

In some embodiments, the antiplatelet agent is atopaxar.

In some embodiments, the antiplatelet agent is cilostazol.

In some embodiments, the antiplatelet agent is prostaglandin E1.

In some embodiments, the antiplatelet agent is epoprostenol.

In some embodiments, the antiplatelet agent is dipyridamole.

In some embodiments, the antiplatelet agent is treprostinil sodium.

In some embodiments, the antiplatelet agent is sarpogrelate.

In some embodiments, the antiplatelet agent is a supplement.

In some embodiments, the antiplatelet agent is an herbal supplement.

In some embodiments, rehydrating the composition comprising platelets orplatelet derivatives comprises adding to the platelets an aqueousliquid. In some embodiments, the aqueous liquid is water. In someembodiments, the aqueous liquid is an aqueous solution (e.g., a buffer).In some embodiments, the aqueous liquid is a saline solution. In someembodiments, the aqueous liquid is a suspension.

In some embodiments, the rehydrated platelets or platelet derivatives(e.g., thrombosomes) have coagulation factor levels showing allindividual factors (e.g., Factors VII, VIII and IX) associated withblood clotting at 40 international units (IU) or greater.

In some embodiments, the platelets such as lyophilized platelets orplatelet derivatives (e.g., thrombosomes) have less than about 10%, suchas less than about 8%, such as less than about 6%, such as less thanabout 4%, such as less than about 2%, such as less than about 0.5%crosslinking of platelet membranes via proteins and/or lipids present onthe membranes. In some embodiments, the rehydrated platelets or plateletderivatives (e.g., thrombosomes), have less than about 10%, such as lessthan about 8%, such as less than about 6%, such as less than about 4%,such as less than about 2%, such as less than about 0.5% crosslinking ofplatelet membranes via proteins and/or lipids present on the membranes.

In some embodiments, the platelets such as lyophilized platelets orplatelet derivatives (e.g., thrombosomes) have a particle size (e.g.,diameter, max dimension) of at least about 0.2 μm (e.g., at least about0.3 μm, at least about 0.4 μm, at least about 0.5 μm, at least about 0.6μm, at least about 0.7 μm, at least about 0.8 μm, at least about 0.9 μm,at least about 1.0 μm, at least about 1.2 μm, at least about 1.5 μm, atleast about 2.0 μm, at least about 2.5 μm, or at least about 5.0 μm). Insome embodiments, the particle size is less than about 5.0 μm (e.g.,less than about 2.5 μm, less than about 2.0 μm, less than about 1.5 μm,less than about 1.0 μm, less than about 0.9 μm, less than about 0.8 μm,less than about 0.7 μm, less than about 0.6 μm, less than about 0.5 μm,less than about 0.4 μm, or less than about 0.3 μm). In some embodiments,the particle size is from about 0.3 μm to about 5.0 μm (e.g., from about0.4 μm to about 4.0 μm, from about 0.5 μm to about 2.5 μm, from about0.6 μm to about 2.0 μm, from about 0.7 μm to about 1.0 μm, from about0.5 μm to about 0.9 μm, or from about 0.6 μm to about 0.8 μm).

In some embodiments, at least 50% (e.g., at least about 55%, at leastabout 60%, at least about 65%, at least about 70%, at least about 75%,at least about 80%, at least about 85%, at least about 90%, at leastabout 95%, or at least about 99%) of platelets such as lyophilizedplatelets or platelet derivatives (e.g., thrombosomes), have a particlesize in the range of about 0.3 μm to about 5.0 μm (e.g., from about 0.4μm to about 4.0 μm, from about 0.5 μm to about 2.5 μm, from about 0.6 μmto about 2.0 μm, from about 0.7 μm to about 1.0 μm, from about 0.5 μm toabout 0.9 μm, or from about 0.6 μm to about 0.8 μm). In someembodiments, at most 99% (e.g., at most about 95%, at most about 80%, atmost about 75%, at most about 70%, at most about 65%, at most about 60%,at most about 55%, or at most about 50%) of the platelets such aslyophilized platelets or platelet derivatives (e.g., thrombosomes), arein the range of about 0.3 μm to about 5.0 μm (e.g., from about 0.4 μm toabout 4.0 μm, from about 0.5 μm to about 2.5 μm, from about 0.6 μm toabout 2.0 μm, from about 0.7 μm to about 1.0 μm, from about 0.5 μm toabout 0.9 μm, or from about 0.6 μm to about 0.8 μm). In someembodiments, about 50% to about 99% (e.g., about 55% to about 95%, about60% to about 90%, about 65% to about 85, about 70% to about 80%) of theplatelets or platelet derivatives (e.g., thrombosomes) are in the rangeof about 0.3 μm to about 5.0 μm (e.g., from about 0.4 μm to about 4.0μm, from about 0.5 μm to about 2.5 μm, from about 0.6 μm to about 2.0μm, from about 0.7 μm to about 1.0 μm, from about 0.5 μm to about 0.9μm, or from about 0.6 μm to about 0.8 μm).

In some embodiments, platelets are isolated, for example in a liquidmedium, prior to treating a subject.

In some embodiments, platelets are donor-derived platelets. In someembodiments, platelets are obtained by a process that comprises anapheresis step. In some embodiments, platelets are pooled platelets.

In some embodiments, platelets are pooled from a plurality of donors.Such platelets pooled from a plurality of donors may be also referredherein to as pooled platelets. In some embodiments, the donors are morethan 5, such as more than 10, such as more than 20, such as more than50, such as up to about 100 donors. In some embodiments, the donors arefrom about 5 to about 100, such as from about 10 to about 50, such asfrom about 20 to about 40, such as from about 25 to about 35. Pooledplatelets can be used to make any of the compositions described herein.

In some embodiments, platelets are derived in vitro. In someembodiments, platelets are derived or prepared in a culture. In someembodiments, preparing the platelets comprises deriving or growing theplatelets from a culture of megakaryocytes. In some embodiments,preparing the platelets comprises deriving or growing the platelets (ormegakaryocytes) from a culture of human pluripotent stem cells (PCSs),including embryonic stem cells (ESCs) and/or induced pluripotent stemcells (iPSCs).

Accordingly, in some embodiments, platelets are prepared prior totreating a subject as described herein. In some embodiments, theplatelets are lyophilized. In some embodiments, the platelets arecryopreserved.

In some embodiments, the platelets or pooled platelets may be acidifiedto a pH of about 6.0 to about 7.4 prior to the incubation with theincubating agent. In some embodiments, the method comprises acidifyingthe platelets to a pH of about 6.5 to about 6.9. In some embodiments,the method comprises acidifying the platelets to a pH of about 6.6 toabout 6.8. In some embodiments, the acidifying comprises adding to thepooled platelets a solution comprising Acid Citrate Dextrose (ACD).

In some embodiments, the platelets are isolated prior to the incubationwith the incubating agent. In some embodiments, the method furthercomprises isolating platelets by using centrifugation. In someembodiments, the centrifugation occurs at a relative centrifugal force(RCF) of about 1000×g to about 2000×g. In some embodiments, thecentrifugation occurs at relative centrifugal force (RCF) of about1300×g to about 1800×g. In some embodiments, the centrifugation occursat relative centrifugal force (RCF) of about 1500×g. In someembodiments, the centrifugation occurs for about 1 minute to about 60minutes. In some embodiments, the centrifugation occurs for about 10minutes to about 30 minutes. In some embodiments, the centrifugationoccurs for about 30 minutes.

An incubating agent can include any appropriate components. In someembodiments, the incubating agent may comprise a liquid medium. In someembodiments the incubating agent may comprise one or more salts selectedfrom phosphate salts, sodium salts, potassium salts, calcium salts,magnesium salts, and any other salt that can be found in blood or bloodproducts, or that is known to be useful in drying platelets, or anycombination of two or more of these.

In some embodiments, the incubating agent comprises one or more salts,such as phosphate salts, sodium salts, potassium salts, calcium salts,magnesium salts, and any other salt that can be found in blood or bloodproducts. Exemplary salts include sodium chloride (NaCl), potassiumchloride (KCl), and combinations thereof. In some embodiments, theincubating agent includes from about 0.5 mM to about 100 mM of the oneor more salts. In some embodiments, the incubating agent includes fromabout 0.5 mM to about 100 mM (e.g., about 0.5 to about 2 mM, about 2 mMto about 90 mM, about 2 mM to about 6 mM, about 50 mM to about 100 mM,about 60 mM to about 90 mM, about 70 to about 85 mM) about of the one ormore salts. In some embodiments, the incubating agent includes about 5mM, about 60 mM, about 65 mM, about 70 mM, about 75 mM, or about 80 mMof the one or more salts. In some embodiments, the incubating agentcomprises one or more salts selected from calcium salts, magnesiumsalts, and a combination of the two, in a concentration of about 0.5 mMto about 2 mM.

Preferably, these salts are present in the composition comprisingplatelets or platelet derivatives, such as freeze-dried platelets, at anamount that is about the same as is found in whole blood.

In some embodiments, the incubating agent further comprises a carrierprotein. In some embodiments, the carrier protein comprises human serumalbumin, bovine serum albumin, or a combination thereof. In someembodiments, the carrier protein is present in an amount of about 0.05%to about 1.0% (w/v).

The incubating agent may be any buffer that is non-toxic to theplatelets and provides adequate buffering capacity to the solution atthe temperatures at which the solution will be exposed during theprocess provided herein. Thus, the buffer may comprise any of the knownbiologically compatible buffers available commercially, such asphosphate buffers, such as phosphate buffered saline (PBS),bicarbonate/carbonic acid, such as sodium-bicarbonate buffer,N-2-hydroxyethylpiperazine-N′-2-ethanesulfonic acid (HEPES), andtris-based buffers, such as tris-buffered saline (TB S). Likewise, itmay comprise one or more of the following buffers:propane-1,2,3-tricarboxylic (tricarballylic); benzenepentacarboxylic;maleic; 2,2-dimethylsuccinic; EDTA; 3,3-dimethylglutaric;bis(2-hydroxyethyl)imino-tris(hydroxymethyl)-methane (BIS-TRIS);benzenehexacarboxylic (mellitic); N-(2-acetamido)imino-diacetic acid(ADA); butane-1,2,3,4-tetracarboxylic; pyrophosphoric;1,1-cyclopentanediacetic (3,3 tetramethylene-glutaric acid);piperazine-1,4-bis-(2-ethanesulfonic acid) (PIPES);N-(2-acetamido)-2-amnoethanesulfonic acid (ACES);1,1-cyclohexanediacetic; 3,6-endomethylene-1,2,3,6-tetrahydrophthalicacid (EMTA; ENDCA); imidazole; 2-(aminoethyl)trimethylammonium chloride(CHOLAMINE); N,N-bis(2-hydroxyethyl)-2-aminoethanesulfonic acid (BES);2-methylpropane-1,2,3-triscarboxylic (beta-methyl tricarballylic);2-(N-morpholino)propane-sulfonic acid (MOPS); phosphoric; andN-tris(hydroxymethyl)methyl-2-amminoethane sulfonic acid (TES). In someembodiments, the incubating agent includes one or more buffers, e.g.,N-2-hydroxyethylpiperazine-N′-2-ethanesulfonic acid (HEPES), orsodium-bicarbonate (NaHCO₃). In some embodiments, the incubating agentincludes from about 5 to about 100 mM of the one or more buffers. Insome embodiments, the incubating agent includes from about 5 to about 50mM (e.g., from about 5 mM to about 40 mM, from about 8 mM to about 30mM, about 10 mM to about 25 mM) about of the one or more buffers. Insome embodiments, the incubating agent includes about 10 mM, about 20mM, about 25 mM, or about 30 mM of the one or more buffers.

In some embodiments, the incubating agent includes one or moresaccharides, such as monosaccharides and disaccharides, includingsucrose, maltose, trehalose, glucose, mannose, dextrose, and xylose. Insome embodiments, the saccharide is a monosaccharide. In someembodiments, the saccharide is a disaccharide. In some embodiments, thesaccharide comprises a monosaccharide, a disaccharide, or a combinationthereof. In some embodiments, the saccharide is a non-reducingdisaccharide. In some embodiments, the saccharide comprises sucrose,maltose, trehalose, glucose (e.g., dextrose), mannose, or xylose. Insome embodiments, the saccharide comprises trehalose. In someembodiments, the incubating agent comprises a starch. In someembodiments, the incubating agent includes polysucrose, a polymer ofsucrose and epichlorohydrin. In some embodiments, the incubating agentincludes from about 10 mM to about 1,000 mM of the one or moresaccharides. In some embodiments, the incubating agent includes fromabout 50 to about 500 mM of the one or more saccharides. In embodiments,one or more saccharides is present in an amount of from 10 mM 10 to 500mM. In some embodiments, one or more saccharides is present in an amountof from 50 mM to 200 mM. In embodiments, one or more saccharides ispresent in an amount from 100 mM to 150 mM. In some embodiments, the oneor more saccharides is the lyophilizing agent; for example, in someembodiments, the lyophilizing agent comprises trehalose, polysucrose, ora combination thereof.

In some embodiments the composition comprising platelets or plateletderivatives, (e.g., thrombosomes), may comprise one or more of water ora saline solution. In some embodiments the composition comprisingplatelets or platelet derivatives, such as freeze-dried platelets, maycomprise DMSO.

In some embodiments, the incubating agent comprises an organic solvent,such as an alcohol (e.g., ethanol). In such an incubating agent, theamount of solvent can range from 0.1% to 5.0% (v/v). In someembodiments, the organic solvent can range from about 0.1% (v/v) toabout 5.0% (v/v), such as from about 0.3% (v/v) to about 3.0% (v/v), orfrom about 0.5% (v/v) to about 2% (v/v).

In some embodiments, suitable organic solvents include, but are notlimited to alcohols, esters, ketones, ethers, halogenated solvents,hydrocarbons, nitriles, glycols, alkyl nitrates, water or mixturesthereof. In some embodiments, suitable organic solvents includes, butare not limited to methanol, ethanol, n-propanol, isopropanol, aceticacid, acetone, methyl ethyl ketone, methyl isobutyl ketone, methylacetate, ethyl acetate, isopropyl acetate, tetrahydrofuran, isopropylether (IPE), tert-butyl methyl ether, dioxane (e.g., 1,4-dioxane),acetonitrile, propionitrile, methylene chloride, chloroform, toluene,anisole, cyclohexane, hexane, heptane, ethylene glycol, nitromethane,dimethylformamide, dimethyl sulfoxide, N-methyl pyrrolidone,dimethylacetamide, and combinations thereof. In some embodiments theorganic solvent is selected from the group consisting of ethanol, aceticacid, acetone, acetonitrile, dimethylformamide, dimethyl sulfoxide(DMSO), dioxane, methanol, n-propanol, isopropanol, tetrahydrofuran(THF), N-methyl pyrrolidone, dimethylacetamide (DMAC), or combinationsthereof. In some embodiments, the organic solvent comprises ethanol,DMSO, or a combination thereof. The presence of organic solvents, suchas ethanol, can be beneficial in the processing of platelets, plateletderivatives, or thrombosomes (e.g., freeze-dried platelet derivatives).

In some embodiments the incubating agent is incubated into the plateletsin the presence of an aqueous medium. In some embodiments the incubatingagent is incubated in the presence of a medium comprising DMSO.

In some embodiments, one or more other components may be incubated inthe platelets. Exemplary components may include Prostaglandin E1 orProstacyclin and or EDTA/EGTA to prevent platelet aggregation andactivation during the incubating process.

Non-limiting examples of incubating agent compositions that may be usedare shown in Tables 1-5.

TABLE 1 Buffer Concentration (mM unless otherwise Component specified)NaCl  75.0 KCl  4.8 HEPES  9.5 NaHCO₃  12.0 Dextrose   3 Trehalose 100Ethanol   1% (optional) (v/v)

TABLE 2 Buffer A Concentration (mM unless specified Component otherwise)CaCl₂ 1.8 MgCl₂ 1.1 KCl 2.7 NaCl 137 NaH₂PO₄ 0.4 HEPES 10 D-glucose 5.6pH 6.5

TABLE 3 Buffer B Concentration (mM unless otherwise Component specified)Buffer and Salts Table 4 (below) BSA 0.35% Dextrose 5 pH 7.4

-   -   Table 3. Buffer B can be used when incubating platelets, e.g.,        for flow cytometry. Such an incubation can be done at room        temperature in the dark. Albumin is an optional component of        Buffer B.

TABLE 4 Concentration of HEPES and of Salts in Buffer B Concentration(mM unless otherwise Component specified) HEPES 25 NaCl 119 KCl 5 CaCl₂2 MgCl₂ 2 glucose 6 g/l

Table 4 is another exemplary incubating agent. The pH can be adjusted to7.4 with NaOH. Albumin is an optional component of Buffer B.

TABLE 5 Tyrode's HEPES Buffer (plus PGE1) Concentration Component (mM)CaCl₂ 1.8 MgCl₂ 1.1 KCl 2.7 NaCl 137 NaH₂PO₄ 0.4 HEPES 10 D-glucose 5.6pH 6.5 Prostaglandin E1 1 μg/ml (PGE1)

Table 5 is another exemplary incubating agent.

In some embodiments, platelets (e.g., apheresis platelets, plateletsisolated from whole blood, pooled platelets, or a combination thereof)are incubated with the incubating agent for different durations at or atdifferent temperatures from 15-45° C., or about 37° C.

In some embodiments, platelets (e.g., apheresis platelets, plateletsisolated from whole blood, pooled platelets, or a combination thereof)form a suspension in an incubating agent comprising a liquid medium at aconcentration from 10,000 platelets/μL to 10,000,000 platelets/μL, suchas 50,000 platelets/μL to 2,000,000 platelets/μL, such as 100,000platelets/μL to 500,000 platelets/μL, such as 150,000 platelets/μL to300,000 platelets/μL, such as 200,000 platelets/μL.

The platelets (e.g., apheresis platelets, platelets isolated from wholeblood, pooled platelets, or a combination thereof) may be incubated withthe incubating agent for different durations, such as, for example, forat least about 5 minutes (mins) (e.g., at least about 20 mins, about 30mins, about 1 hour (hr), about 2 hrs, about 3 hrs, about 4 hrs, about 5hrs, about 6 hrs, about 7 hrs, about 8 hrs, about 9 hrs, about 10 hrs,about 12 hrs, about 16 hrs, about 20 hrs, about 24 hrs, about 30 hrs,about 36 hrs, about 42 hrs, about 48 hrs, or at least about 48 hrs. Insome embodiments, the platelets may be incubated with the incubatingagent for no more than about 48 hrs (e.g., no more than about 20 mins,about 30 mins, about 1 hour (hr), about 2 hrs, about 3 hrs, about 4 hrs,about 5 hrs, about 6 hrs, about 7 hrs, about 8 hrs, about 9 hrs, about10 hrs, about 12 hrs, about 16 hrs, about 20 hrs, about 24 hrs, about 30hrs, about 36 hrs, or no more than about 42 hrs). In some embodiments,the platelets may be incubated with the incubating agent for from about10 mins to about 48 hours (e.g., from about 20 mins to about 36 hrs,from about 30 mins to about 24 hrs, from about 1 hr to about 20 hrs,from about 2 hrs to about 16 hours, from about 10 mins to about 24hours, from about 20 mins to about 12 hours, from about 30 mins to about10 hrs, or from about 1 hr to about 6 hrs. In some embodiments, theplatelets, the platelet derivatives, or the thrombosomes are incubatedwith the incubating agent for a period of time of 5 minutes to 48 hours,such as 10 minutes to 24 hours, such as 20 minutes to 12 hours, such as30 minutes to 6 hours, such as 1 hour minutes to 3 hours, such as about2 hours.

In some embodiments, the platelets (e.g., apheresis platelets, plateletsisolated from whole blood, pooled platelets, or a combination thereof)are incubated with the incubating agents at different temperatures. Inembodiments, incubation is conducted at 37° C. In certain embodiments,incubation is performed at 4° C. to 45° C., such as 15° C. to 42° C. Forexample, in embodiments, incubation is performed at 35° C. to 40° C.(e.g., 37° C.) for 110 to 130 (e.g., 120) minutes and for as long as24-48 hours. In some embodiments, the platelets are incubated with theincubating agent for different durations as disclosed herein, and attemperatures from 15-45° C., or about 37° C.

In some embodiments, platelets (e.g., apheresis platelets, plateletsisolated from whole blood, pooled platelets, or a combination thereof)are loaded with one or more active agents. In some embodiments, theplatelets can be loaded with an anti-fibrinolytic agent. Non-limitingexamples of anti-fibrinolytic agents include ε-aminocaproic acid (EACA),tranexamic acid, aprotinin, aminomethylbenzoic acid, and fibrinogen.

Loading platelets (e.g., apheresis platelets, platelets isolated fromwhole blood, pooled platelets, or a combination thereof) with an activeagent (e.g., an anti-fibrinolytic agent) can be performed by anyappropriate method. See, for example, PCT Publication Nos.WO2020113090A1, WO2020113101A1, WO2020113035A1, and WO2020112963A1.Generally, the loading includes contacting the platelets with theanti-fibrinolytic agent. In some embodiments, the loading can beperformed by combining the active agent with the incubating agent. Insome embodiments, the loading can be performed in a separate step fromthe incubating step. For example, the loading can be performed in a stepprior to the incubation step. In some such embodiments, the active agentcan be supplied to the platelets as a solution or suspension in any ofthe incubation agents described herein, which may or may not be the sameas the incubating agent used in the incubating step. In someembodiments, the loading step can be performed during the incubationstep. In some such embodiments, the active agent can be added to theincubation agent (e.g., as a solid or in a solution or suspension)during the incubation). In some embodiments, the loading step can beperformed in a step following the incubation step. In some suchembodiments, be supplied to the platelets as a solution or suspension inany of the incubation agents described herein, which may or may not bethe same as the incubating agent used in the incubating step.

An active agent can be applied to the platelets in any appropriateconcentration. In some embodiments, an active agent can be applied tothe platelets (e.g., as part of the incubating agent or another solutionor suspension) in a concentration of about 1 μM to about 100 mM (e.g.,about 1 μM to about 10 μm, about 1 μM to about 50 about 1 μM to about100 about 1 μM to about 500 about 1 μM to about 1 mM, about 1 μM toabout 10 mM, about 1 μM to about 25 mM, about 1 μM to about 50 mM, about1 μM to about 75 mM, about 10 μM to about 100 mM, about 50 μM to about100 mM, about 100 μM to about 100 mM, about 500 μM to about 100 mM,about 1 mM to about 100 mM, about 10 mM to about 100 mM, about 25 mM toabout 100 mM, about 50 mM to about 100 mM, about 75 mM to about 100 mM,about 10 μM to about 100 mM, about 200 μM to about 1 mM, about 800 μM toabout 900 about 400 μM to about 800 about 500 μM to about 700 about 600about 5 mM to about 85 mM, about 20 mM to about 90 mM, about 25 mM toabout 75 mM, about 30 mM to about 90 mM, about 35 mM to about 65 mM,about 40 mM to about 60 mM, about 50 mM to about 60 mM, about 40 mM toabout 70 mM, about 45 mM to about 55 mM, or about 50 mM).

In some embodiments, the method further comprises drying the platelets.In some embodiments, the drying step comprises lyophilizing theplatelets. In some embodiments, the drying step comprises freeze-dryingthe platelets. In some embodiments, the method further comprisesrehydrating the platelets obtained from the drying step.

In some embodiments, the platelets are cold stored, cryopreserved, orlyophilized (e.g., to produce thrombosomes) prior to use in therapy orin functional assays.

Any known technique for drying platelets can be used in accordance withthe present disclosure, as long as the technique can achieve a finalresidual moisture content of less than 5%. Preferably, the techniqueachieves a final residual moisture content of less than 2%, such as 1%,0.5%, or 0.1%. Non-limiting examples of suitable techniques arefreeze-drying (lyophilization) and spray-drying. A suitablelyophilization method is presented in Table A. Additional exemplarylyophilization methods can be found in U.S. Pat. Nos. 7,811,558,8,486,617, and 8,097,403. An exemplary spray-drying method includes:combining nitrogen, as a drying gas, with a incubating agent accordingto the present disclosure, then introducing the mixture into GEA MobileMinor spray dryer from GEA Processing Engineering, Inc. (Columbia Md.,USA), which has a Two-Fluid Nozzle configuration, spray drying themixture at an inlet temperature in the range of 150° C. to 190° C., anoutlet temperature in the range of 65° C. to 100° C., an atomic rate inthe range of 0.5 to 2.0 bars, an atomic rate in the range of 5 to 13kg/hr, a nitrogen use in the range of 60 to 100 kg/hr, and a run time of10 to 35 minutes. The final step in spray drying is preferentiallycollecting the dried mixture. The dried composition in some embodimentsis stable for at least six months at temperatures that range from −20°C. or lower to 90° C. or higher.

TABLE A Exemplary Lyophilization Protocol Pressure Step Temp. Set TypeDuration Set Freezing Step F1 −50° C. Ramp Var N/A F2 −50° C. Hold 3 HrsN/A Vacuum Pulldown F3 −50º Hold Var N/A Primary Dry P1 −40º Hold 1.5Hrs 0 mT P2 −35º Ramp 2 Hrs 0 mT P3 −25º Ramp 2 Hrs 0 mT P4 −17° C. Ramp2 Hrs 0 mT P5   0° C. Ramp 1.5 Hrs 0 mT P6  27° C. Ramp 1.5 Hrs 0 mT P7 27° C. Hold 16 Hrs 0 mT Secondary Dry S1  27° C. Hold >8 Hrs 0 mT

In some embodiments, the step of drying the platelets that are obtainedas disclosed herein, such as the step of freeze-drying the plateletsthat are obtained as disclosed herein, comprises incubating theplatelets with a lyophilizing agent (e.g., a non-reducing disaccharide).Accordingly, in some embodiments, the methods for preparing plateletsfurther comprise incubating the platelets with a lyophilizing agent. Insome embodiments the lyophilizing agent is a saccharide. In someembodiments the saccharide is a disaccharide, such as a non-reducingdisaccharide.

In some embodiments, the platelets are incubated with a lyophilizingagent for a sufficient amount of time and at a suitable temperature toincubate the platelets with the lyophilizing agent. Non-limitingexamples of suitable lyophilizing agents are saccharides, such asmonosaccharides and disaccharides, including sucrose, maltose,trehalose, glucose (e.g., dextrose), mannose, and xylose. In someembodiments, non-limiting examples of lyophilizing agents include serumalbumin, dextran, polyvinyl pyrrolidone (PVP), starch, and hydroxyethylstarch (HES). In some embodiments, exemplary lyophilizing agents caninclude a high molecular weight polymer. By “high molecular weight” itmeans a polymer having an average molecular weight of about or above 70kDa and up to 1,000,000 kDa. Non-limiting examples are polymers ofsucrose and epichlorohydrin (e.g., polysucrose). In some embodiments,the lyophilizing agent is polysucrose. Although any amount of highmolecular weight polymer can be used as a lyophilizing agent, it ispreferred that an amount be used that achieves a final concentration ofabout 3% to 10% (w/v), such as 3% to 7%, for example 6%.

An exemplary saccharide for use in the compositions disclosed herein istrehalose. Regardless of the identity of the saccharide, it can bepresent in the composition in any suitable amount. For example, it canbe present in an amount of 1 mM to 1 M. In embodiments, it is present inan amount of from 10 mM 10 to 500 mM. In some embodiments, it is presentin an amount of from 20 mM to 200 mM. In embodiments, it is present inan amount from 40 mM to 100 mM. In various embodiments, the saccharideis present in different specific concentrations within the rangesrecited above, and one of skill in the art can immediately understandthe various concentrations without the need to specifically recite eachherein. Where more than one saccharide is present in the composition,each saccharide can be present in an amount according to the ranges andparticular concentrations recited above.

Within the process provided herein for making the compositions providedherein, addition of the lyophilizing agent can be the last step prior todrying. However, in some embodiments, the lyophilizing agent is added atthe same time or before other components of the composition, such as asalt, a buffer, optionally a cryoprotectant, or other components. Insome embodiments, the lyophilizing agent is added to the incubatingagent, thoroughly mixed to form a drying solution, dispensed into adrying vessel (e.g., a glass or plastic serum vial, a lyophilizationbag), and subjected to conditions that allow for drying of the solutionto form a dried composition.

The step of incubating the platelets with a cryoprotectant can includeincubating the platelets for a time suitable for loading, as long as thetime, taken in conjunction with the temperature, is sufficient for thecryoprotectant to come into contact with the platelets and, preferably,be incorporated, at least to some extent, into the platelets. Inembodiments, incubation is carried out for about 1 minute to about 180minutes or longer.

The step of incubating the platelets with a cryoprotectant can includeincubating the platelets and the cryoprotectant at a temperature that,when selected in conjunction with the amount of time allotted, issuitable for incubating. In general, the composition is incubated at atemperature above freezing for at least a sufficient time for thecryoprotectant to come into contact with the platelets. In embodiments,incubation is conducted at 37° C. In certain embodiments, incubation isperformed at 20° C. to 42° C. For example, in embodiments, incubation isperformed at 35° C. to 40° C. (e.g., 37° C.) for 110 to 130 (e.g., 120)minutes.

In various embodiments, the lyophilization bag is a gas-permeable bagconfigured to allow gases to pass through at least a portion or allportions of the bag during the processing. The gas-permeable bag canallow for the exchange of gas within the interior of the bag withatmospheric gas present in the surrounding environment. Thegas-permeable bag can be permeable to gases, such as oxygen, nitrogen,water, air, hydrogen, and carbon dioxide, allowing gas exchange to occurin the compositions provided herein. In some embodiments, thegas-permeable bag allows for the removal of some of the carbon dioxidepresent within an interior of the bag by allowing the carbon dioxide topermeate through its wall. In some embodiments, the release of carbondioxide from the bag can be advantageous to maintaining a desired pHlevel of the composition contained within the bag.

In some embodiments, the container of the process herein is agas-permeable container that is closed or sealed. In some embodiments,the container is a container that is closed or sealed and a portion ofwhich is gas-permeable. In some embodiments, the surface area of agas-permeable portion of a closed or sealed container (e.g., bag)relative to the volume of the product being contained in the container(hereinafter referred to as the “SA/V ratio”) can be adjusted to improvepH maintenance of the compositions provided herein. For example, in someembodiments, the SA/V ratio of the container can be at least about 2.0cm²/mL (e.g., at least about 2.1 cm²/mL, at least about 2.2 cm²/mL, atleast about 2.3 cm²/mL, at least about 2.4 cm²/mL, at least about 2.5cm²/mL, at least about 2.6 cm²/mL, at least about 2.7 cm²/mL, at leastabout 2.8 cm²/mL, at least about 2.9 cm²/mL, at least about 3.0 cm²/mL,at least about 3.1 cm²/mL, at least about 3.2 cm²/mL, at least about 3.3cm²/mL, at least about 3.4 cm²/mL, at least about 3.5 cm²/mL, at leastabout 3.6 cm²/mL, at least about 3.7 cm²/mL, at least about 3.8 cm²/mL,at least about 3.9 cm²/mL, at least about 4.0 cm²/mL, at least about 4.1cm²/mL, at least about 4.2 cm²/mL, at least about 4.3 cm²/mL, at leastabout 4.4 cm²/mL, at least about 4.5 cm²/mL, at least about 4.6 cm²/mL,at least about 4.7 cm²/mL, at least about 4.8 cm²/mL, at least about 4.9cm²/mL, or at least about 5.0 cm²/mL. In some embodiments, the SA/Vratio of the container can be at most about 10.0 cm²/mL (e.g., at mostabout 9.9 cm²/mL, at most about 9.8 cm²/mL, at most about 9.7 cm²/mL, atmost about 9.6 cm²/mL, at most about 9.5 cm²/mL, at most about 9.4cm²/mL, at most about 9.3 cm²/mL, at most about 9.2 cm²/mL, at mostabout 9.1 cm²/mL, at most about 9.0 cm²/mL, at most about 8.9 cm²/mL, atmost about 8.8 cm²/mL, at most about 8.7 cm²/mL, at most about 8.6,cm²/mL at most about 8.5 cm²/mL, at most about 8.4 cm²/mL, at most about8.3 cm²/mL, at most about 8.2 cm²/mL, at most about 8.1 cm²/mL, at mostabout 8.0 cm²/mL, at most about 7.9 cm²/mL, at most about 7.8 cm²/mL, atmost about 7.7 cm²/mL, at most about 7.6 cm²/mL, at most about 7.5cm²/mL, at most about 7.4 cm²/mL, at most about 7.3 cm²/mL, at mostabout 7.2 cm²/mL, at most about 7.1 cm²/mL, at most about 6.9 cm²/mL, atmost about 6.8 cm²/mL, at most about 6.7 cm²/mL, at most about 6.6cm²/mL, at most about 6.5 cm²/mL, at most about 6.4 cm²/mL, at mostabout 6.3 cm²/mL, at most about 6.2 cm²/mL, at most about 6.1 cm²/mL, atmost about 6.0 cm²/mL, at most about 5.9 cm²/mL, at most about 5.8cm²/mL, at most about 5.7 cm²/mL, at most about 5.6 cm²/mL, at mostabout 5.5 cm²/mL, at most about 5.4 cm²/mL, at most about 5.3 cm²/mL, atmost about 5.2 cm²/mL, at most about 5.1 cm²/mL, at most about 5.0cm²/mL, at most about 4.9 cm²/mL, at most about 4.8 cm²/mL, at mostabout 4.7 cm²/mL, at most about 4.6 cm²/mL, at most about 4.5 cm²/mL, atmost about 4.4 cm²/mL, at most about 4.3 cm²/mL, at most about 4.2cm²/mL, at most about 4.1 cm²/mL, or at most about 4.0 cm²/mL. In someembodiments, the SA/V ratio of the container can range from about 2.0 toabout 10.0 cm²/mL (e.g., from about 2.1 cm²/mL to about 9.9 cm²/mL, fromabout 2.2 cm²/mL to about 9.8 cm²/mL, from about 2.3 cm²/mL to about 9.7cm²/mL, from about 2.4 cm²/mL to about 9.6 cm²/mL, from about 2.5 cm²/mLto about 9.5 cm²/mL, from about 2.6 cm²/mL to about 9.4 cm²/mL, fromabout 2.7 cm²/mL to about 9.3 cm²/mL, from about 2.8 cm²/mL to about 9.2cm²/mL, from about 2.9 cm²/mL to about 9.1 cm²/mL, from about 3.0 cm²/mLto about 9.0 cm²/mL, from about 3.1 cm²/mL to about 8.9 cm²/mL, fromabout 3.2 cm²/mL to about 8.8 cm²/mL, from about 3.3 cm²/mL to about 8.7cm²/mL, from about 3.4 cm²/mL to about 8.6 cm²/mL, from about 3.5 cm²/mLto about 8.5 cm²/mL, from about 3.6 cm²/mL to about 8.4 cm²/mL, fromabout 3.7 cm²/mL to about 8.3 cm²/mL, from about 3.8 cm²/mL to about 8.2cm²/mL, from about 3.9 cm²/mL to about 8.1 cm²/mL, from about 4.0 cm²/mLto about 8.0 cm²/mL, from about 4.1 cm²/mL to about 7.9 cm²/mL, fromabout 4.2 cm²/mL to about 7.8 cm²/mL, from about 4.3 cm²/mL to about 7.7cm²/mL, from about 4.4 cm²/mL to about 7.6 cm²/mL, from about 4.5 cm²/mLto about 7.5 cm²/mL, from about 4.6 cm²/mL to about 7.4 cm²/mL, fromabout 4.7 cm²/mL to about 7.3 cm²/mL, from about 4.8 cm²/mL to about 7.2cm²/mL, from about 4.9 cm²/mL to about 7.1 cm²/mL, from about 5.0 cm²/mLto about 6.9 cm²/mL, from about 5.1 cm²/mL to about 6.8 cm²/mL, fromabout 5.2 cm²/mL to about 6.7 cm²/mL, from about 5.3 cm²/mL to about 6.6cm²/mL, from about 5.4 cm²/mL to about 6.5 cm²/mL, from about 5.5 cm²/mLto about 6.4 cm²/mL, from about 5.6 cm²/mL to about 6.3 cm²/mL, fromabout 5.7 cm²/mL to about 6.2 cm²/mL, or from about 5.8 cm²/mL to about6.1 cm²/mL.

Gas-permeable closed containers (e.g., bags) or portions thereof can bemade of one or more various gas-permeable materials. In someembodiments, the gas-permeable bag can be made of one or more polymersincluding fluoropolymers (such as polytetrafluoroethylene (PTFE) andperfluoroalkoxy (PFA) polymers), polyolefins (such as low-densitypolyethylene (LDPE), high-density polyethylene (HDPE)), fluorinatedethylene propylene (FEP), polystyrene, polyvinylchloride (PVC),silicone, and any combinations thereof.

In some embodiments, dried platelets or platelet derivatives (e.g.,thrombosomes) can undergo heat treatment. Heating can be performed at atemperature above about 25° C. (e.g., greater than about 40° C., 50° C.,60° C., 70° C., 80° C. or higher). In some embodiments, heating isconducted between about 70° C. and about 85° C. (e.g., between about 75°C. and about 85° C., or at about 75° C. or 80° C.). The temperature forheating can be selected in conjunction with the length of time thatheating is to be performed. Although any suitable time can be used,typically, the lyophilized platelets are heated for at least 1 hour, butnot more than 36 hours. Thus, in embodiments, heating is performed forat least 2 hours, at least 6 hours, at least 12 hours, at least 18hours, at least 20 hours, at least 24 hours, or at least 30 hours. Forexample, the lyophilized platelets can be heated for 18 hours, 19 hours,20 hours, 21 hours, 22 hours, 23 hours, 24 hours, 25 hours, 26 hours, 27hours, 28 hours, 29 hours, or 30 hours. Non-limiting exemplarycombinations include: heating the dried platelets or plateletderivatives (e.g., thrombosomes) for at least 30 minutes at atemperature higher than 30° C.; heating the dried platelets or plateletderivatives (e.g., thrombosomes) for at least 10 hours at a temperaturehigher than 50° C.; heating the dried platelets or platelet derivatives(e.g., thrombosomes) for at least 18 hours at a temperature higher than75° C.; and heating the dried platelets or platelet derivatives (e.g.,thrombosomes) for 24 hours at 80° C. In some embodiments, heating can beperformed in sealed container, such as a capped vial. In someembodiments, a sealed container be subjected to a vacuum prior toheating. The heat treatment step, particularly in the presence of acryoprotectant such as albumin or polysucrose, has been found to improvethe stability and shelf-life of the freeze-dried platelets. Indeed,advantageous results have been obtained with the particular combinationof serum albumin or polysucrose and a post-lyophilization heat treatmentstep, as compared to those cryoprotectants without a heat treatmentstep. A cryoprotectant (e.g., sucrose) can be present in any appropriateamount (e.g. about 3% to about 10% by mass or by volume of the plateletsor platelet derivatives (e.g., thrombosomes).

In some embodiments, the platelets or platelet derivatives (e.g.,thrombosomes) prepared as disclosed herein by a process comprisingincubation with an incubating agent have a storage stability that is atleast about equal to that of the platelets prior to the incubation.

In some embodiments, the method further comprises cryopreserving theplatelets or platelet derivatives prior to administering the plateletsor platelet derivatives (e.g., with an incubating agent, e.g., anincubating agent described herein).

In some embodiments, the method further comprises drying a compositioncomprising platelets or platelet derivatives, (e.g., with an incubatingagent e.g., an incubating agent described herein) prior to administeringthe platelets or platelet derivatives (e.g., thrombosomes). In someembodiments, the method may further comprise heating the compositionfollowing the drying step. In some embodiments, the method may furthercomprise rehydrating the composition following the freeze-drying step orthe heating step.

In some embodiments, the method further comprises freeze-drying acomposition comprising platelets or platelet derivatives (e.g., with anincubating agent e.g., an incubating agent described herein) prior toadministering the platelets or platelet derivatives (e.g., thrombosomes)In some embodiments, the method may further comprise heating thecomposition following the freeze-drying step. In some embodiments, themethod may further comprise rehydrating the composition following thefreeze-drying step or the heating step.

In some embodiments, the method further comprises cold storing theplatelets, platelet derivatives, or the thrombosomes prior toadministering the platelets, platelet derivatives, or thrombosomes(e.g., with an incubating agent, e.g., an incubating agent describedherein).

Storing conditions include, for example, standard room temperaturestoring (e.g., storing at a temperature ranging from about 20 to about30° C.) or cold storing (e.g., storing at a temperature ranging fromabout 1 to about 10° C.). In some embodiments, the method furthercomprises cryopreserving, freeze-drying, thawing, rehydrating, andcombinations thereof, a composition comprising platelets or plateletderivatives (e.g., thrombosomes) (e.g., with an incubating agent e.g.,an incubating agent described herein) prior to administering theplatelets or platelet derivatives (e.g., thrombosomes). For example, insome embodiments, the method further comprises drying (e.g.,freeze-drying) a composition comprising platelets or plateletderivatives (e.g., with an incubating agent e.g., an incubating agentdescribed herein) (e.g., to form thrombosomes) prior to administeringthe platelets or platelet derivatives (e.g., thrombosomes). In someembodiments, the method may further comprise rehydrating the compositionobtained from the drying step.

In some embodiments, provided herein is composition comprising plateletsor platelet derivatives (e.g., thrombosomes), polysucrose and trehalosemade by the process of obtaining fresh platelets, optionally incubatingthe platelets in DMSO, isolating the platelets by centrifugation,resuspending the platelets in an incubating agent which comprisestrehalose and ethanol thereby forming a first mixture, incubating thefirst mixture, mixing polysucrose with the first mixture, therebyforming a second mixture, and lyophilizing the second mixture to form afreeze dried composition comprising platelets or platelet derivatives(e.g., thrombosomes), polysucrose and trehalose.

In some embodiments, provided herein is a method of making afreeze-dried platelet composition comprising platelets or plateletderivatives (e.g., thrombosomes), polysucrose and trehalose comprisingobtaining fresh platelets, optionally incubating the platelets in DMSO,isolating the platelets by centrifugation, resuspending the platelets ina incubating agent which comprises trehalose and ethanol thereby forminga first mixture, incubating the first mixture, mixing polysucrose withthe first mixture, thereby forming a second mixture, and lyophilizingthe second mixture to form a freeze-dried composition comprisingplatelets or platelet derivatives (e.g., thrombosomes), polysucrose andtrehalose.

In some embodiments, provided herein is a process for makingfreeze-dried platelets, the process comprising incubating isolatedplatelets in the presence of at least one saccharide under the followingconditions: a temperature of from 20° C. to 42° C. for about 10 minutesto about 180 minutes, adding to the platelets at least onecryoprotectant, and lyophilizing the platelets, wherein the processoptionally does not include isolating the platelets between theincubating and adding steps, and optionally wherein the process does notinclude exposing the platelets to a platelet activation inhibitor. Thecryoprotectant can be a polysugar (e.g., polysucrose). The process canfurther include heating the lyophilized platelets at a temperature of70° C. to 80° C. for 8 to 24 hours. The step of adding to the plateletsat least one cryoprotectant can further include exposing the plateletsto ethanol. The step of incubating isolated platelets in the presence ofat least one saccharide can include incubating in the presence of atleast one saccharide. The step of incubating isolated platelets in thepresence of at least one saccharide can include incubating in thepresence of at least one saccharide. The conditions for incubating caninclude incubating for about 100 minutes to about 150 minutes. Theconditions for incubating can include incubating for about 110 minutesto about 130 minutes. The conditions for incubating can includeincubating for about 120 minutes. The conditions for incubating caninclude incubating at 35° C. to 40° C. The conditions for incubating caninclude incubating at 37° C. The conditions for incubating can includeincubating at 35° C. to 40° C. for 110 minutes to 130 minutes. Theconditions for incubating can include incubating at 37° C. for 120minutes. The at least one saccharide can be trehalose, sucrose, or bothtrehalose and sucrose. The at least one saccharide can be trehalose. Theat least one saccharide can be sucrose.

In some embodiments, provided herein is a method of preparingfreeze-dried platelets, the method including providing platelets,suspending the platelets in a salt buffer that includes about 100 mMtrehalose and about 1% (v/v) ethanol to make a first composition,incubating the first composition at about 37° C. for about 2 hours,adding polysucrose (e.g., polysucrose 400) to a final concentration ofabout 6% (w/v) to make a second composition, lyophilizing the secondcomposition to make freeze-dried platelets, and heating the freeze-driedplatelets at 80° C. for 24 hours.

Specific embodiments disclosed herein may be further limited in theclaims using “consisting of” or “consisting essentially of” language.

Exemplary Embodiments

Embodiment 1 is a method of treating a coagulopathy in a subject, themethod comprising administering to the subject in need thereof aneffective amount of a composition comprising platelets or plateletderivatives and an incubating agent comprising one or more salts, abuffer, optionally a cryoprotectant, and optionally an organic solvent.

Embodiment 2 is a method of treating a coagulopathy in a subject, themethod comprising administering to the subject in need thereof aneffective amount of a composition prepared by a process comprisingincubating platelets with an incubating agent comprising one or moresalts, a buffer, optionally a cryoprotectant, and optionally an organicsolvent, to form the composition.

Embodiment 3 is a method of restoring normal hemostasis in a subject,the method comprising administering to the subject in need thereof aneffective amount of a composition comprising platelets or plateletderivatives and an incubating agent comprising one or more salts, abuffer, optionally a cryoprotectant, and optionally an organic solvent.

Embodiment 4 is a method of restoring normal hemostasis in a subject,the method comprising administering to the subject in need thereof aneffective amount of a composition prepared by a process comprisingincubating platelets with an incubating agent comprising one or moresalts, a buffer, optionally a cryoprotectant, and optionally an organicsolvent, to form the composition.

Embodiment 5 is a method of preparing a subject for surgery, the methodcomprising administering to the subject in need thereof an effectiveamount of a composition comprising platelets or platelet derivatives andan incubating agent comprising one or more salts, a buffer, optionally acryoprotectant, and optionally an organic solvent.

Embodiment 6 is a method of preparing a subject for surgery, the methodcomprising administering to the subject in need thereof an effectiveamount of a composition prepared by a process comprising incubatingplatelets with an incubating agent comprising one or more salts, abuffer, optionally a cryoprotectant, and optionally an organic solvent,to form the composition.

Embodiment 7 is the method of any one of embodiments 5-6, wherein thesurgery is an emergency surgery.

Embodiment 8 is the method of any one of embodiments 5-6, wherein thesurgery is a scheduled surgery.

Embodiment 9 is the method of any one of embodiments 1-8, wherein thesubject has been treated or is being treated with an antiplatelet agent.

Embodiment 10 is the method of embodiment 9, wherein treatment with theantiplatelet agent is stopped.

Embodiment 11 is the method of embodiment 9, wherein treatment with theantiplatelet agent is continued.

Embodiment 12 is a method of ameliorating the effects of an antiplateletagent in a subject, the method comprising administering to the subjectin need thereof an effective amount of a composition comprisingplatelets or platelet derivatives and an incubating agent comprising oneor more salts, a buffer, optionally a cryoprotectant, and optionally anorganic solvent.

Embodiment 13 is a method of ameliorating the effects of an antiplateletagent in a subject, the method comprising administering to the subjectin need thereof an effective amount of a composition prepared by aprocess comprising incubating platelets with an incubating agentcomprising one or more salts, a buffer, optionally a cryoprotectant, andoptionally an organic solvent, to form the composition.

Embodiment 14 is the method of embodiment 12 or embodiment 13, whereinthe effects of the antiplatelet agent are the result of an overdose ofthe antiplatelet agent.

Embodiment 15 is the method of any one of embodiments 1-14, wherein thecomposition further comprises an anti-fibrinolytic agent.

Embodiment 16 is the method of embodiment 15, wherein theanti-fibrinolytic agent is selected from the group consisting ofε-aminocaproic acid (EACA), tranexamic acid, aprotinin,aminomethylbenzoic acid, fibrinogen, and a combination thereof.

Embodiment 17 is the method of embodiment 15 or embodiment 16, whereinthe platelets or platelet derivatives are loaded with theanti-fibrinolytic agent.

Embodiment 18 is the method of any one of embodiments 9-16, wherein theantiplatelet agent is selected from the group consisting of aspirin,cangrelor, ticagrelor, clopidogrel, prasugrel, eptifibatide, tirofiban,abciximab, a supplement, and a combination thereof.

Embodiment 19 is the method of any one of embodiments 9-16, wherein theantiplatelet agent is selected from the group consisting of aspirin,cangrelor, ticagrelor, clopidogrel, prasugrel, eptifibatide, tirofiban,abciximab, terutroban, picotamide, elinogrel, ticlopidine, ibuprofen,vorapaxar, atopaxar, and a combination thereof.

Embodiment 20 is the method of any one of embodiments 9-16, wherein theantiplatelet agent is selected from the group consisting of aspirin,cangrelor, ticagrelor, clopidogrel, prasugrel, eptifibatide, tirofiban,abciximab, terutroban, picotamide, elinogrel, ticlopidine, ibuprofen,vorapaxar, atopaxar, cilostazol, prostaglandin E1, epoprostenol,dipyridamole, treprostinil sodium, sarpogrelate, and a combinationthereof.

Embodiment 21 is the method of any one of embodiments 1-20, whereinadministering comprises administering topically.

Embodiment 22 is the method of any one of embodiments 1-20, whereinadministering comprises administering parenterally.

Embodiment 23 is the method of any one of embodiments 1-20, whereinadministering comprises administering intravenously.

Embodiment 24 is the method of any one of embodiments 1-20, whereinadministering comprises administering intramuscularly.

Embodiment 25 is the method of any one of embodiments 1-20, whereinadministering comprises administering intrathecally.

Embodiment 26 is the method of any one of embodiments 1-20, whereinadministering comprises administering subcutaneously.

Embodiment 27 is the method of any one of embodiments 1-20, whereinadministering comprises administering intraperitoneally.

Embodiment 28 is the method of any one of embodiments 1-27, wherein thecomposition is dried prior to the administration step.

Embodiment 29 is the method of embodiment 28, wherein the composition isrehydrated following the drying step.

Embodiment 30 is the method of any one of embodiments 1-28, wherein thecomposition is freeze-dried prior to the administration step.

Embodiment 31 is the method of embodiment 30, wherein the composition isrehydrated following the freeze-drying step.

Embodiment 32 is the method of any one of embodiments 1-31, wherein theincubating agent comprises one or more salts selected from phosphatesalts, sodium salts, potassium salts, calcium salts, magnesium salts,and a combination of two or more thereof.

Embodiment 33 is the method of any one of embodiments 1-32, wherein theincubating agent comprises a carrier protein.

Embodiment 34 is the method of any one of embodiments 1-33, wherein thebuffer comprises HEPES, sodium bicarbonate (NaHCO₃), or a combinationthereof.

Embodiment 35 is the method of any one of embodiments 1-34, wherein thecomposition comprises one or more saccharides.

Embodiment 36 is the method of embodiment 35, wherein the one or moresaccharides comprise trehalose.

Embodiment 37 is the method of embodiment 35 or embodiment 36, whereinthe one or more saccharides comprise polysucrose.

Embodiment 38 is the method of any one of embodiments 35-37, wherein theone or more saccharides comprise dextrose.

Embodiment 39 is the method of any one of embodiments 1-38, wherein thecomposition comprises an organic solvent.

Embodiment 40 is the method of any of embodiments 1-39, wherein theplatelets or platelet derivatives comprise thrombosomes.

EXAMPLES

The results that follow demonstrate the impact of the thrombosomesproduct in an in vitro model of a patient taking antiplatelet drugs.Thrombosomes and other lyophilized platelet products are designed forinfusion into a patient's bloodstream following diagnosis of trauma orhemostatic failure. These drugs utilize multiple forms of plateletinhibition mechanisms which inhibit platelet response to adenosinediphosphate (ADP), arachidonic acid, fibrinogen and von Willebrandfactor binding to name a few. These include drugs like aspirin,clopidogrel, ticagrelor, effient, cangrelor and eptifibatide.

Example 1—P2Y₁₂ Inhibitors

Cangrelor, like clopidogrel, ticagrelor, and prasugurel, blocks theP2Y₁₂ (ADP) receptor on platelets. Cangrelor is used here as arepresentative of this class of drug.

Thrombosomes were prepared consistent with the procedure in Example 4.Transmission light aggregometry and T-TAS® experiments were carried outaccording to Example 4.

The effect of cangrelor on the aggregation of platelets in platelet-richplasma (PRP; taken from humans as whole blood and processed to isolateplatelets in plasma without white blood cells (WBC) or red blood cells(rbc) was evaluated by transmission light aggregometry. Aggregation ofplatelets (platelet rich plasma) in response to agonist-inducedactivation showed complete inhibition of 10 μM adenosine diphosphate(ADP)-induced aggregation by cangrelor at therapeutic concentration of0.5 μM-3.5 μM (FIG. 1 ). All doses of cangrelor investigated completelyeliminated ADP-induced platelet aggregation in PRP.

The effect of cangrelor on platelet occlusion under shear was evaluatedby T-TAS®. Fresh platelet rich plasma (platelet concentration278,000/μL; PRP generally has a platelet concentration of about200,000/μL to about 300,000/μL) stimulated in vitro with 10 ADP occludedearlier under high shear than unstimulated platelets (PRP) as determinedby AR chip (collagen and tissue thromboplastin) using T-TAS® technology(FIG. 2 ). Cangrelor alone (1 μM) did not exhibit inhibition onocclusion, but when combined with ADP (10 μM), platelet adhesion andocclusion was essentially eliminated. These results are furtherillustrated in FIGS. 3 and 4 . Without being bound by any particulartheory, it is believed that this pattern is observed because plateletshave other ADP receptors not blocked by cangrelor that respond to ADPand cause shape change and aggregation where the ADP receptor P2Y12blocking inhibits collagen binding, and, accordingly, the platelets maybind each other due to ADP stimulation but may be prevented from bindingcollagen on the coated chip.

In FIG. 3 , the area under the curve (AUC) values (derived from data inFIG. 2 ; replicates are averaged and plotted once) are indicative of acombined value of how quickly the thrombus happened in time and howsubstantial the thrombus is when it does happen. PRP AUC was increasedwith ADP stimulation. Cangrelor had little effect on AUC value, but whencombined with ADP stimulation, the AUC dropped close to zero.

In FIG. 4 , the time to occlusion of the AR T-TAS® chip with drugtreatment was evaluated. PRP occluded the chip channel at approximately20 minutes, and stimulation of platelets with ADP decreased that time.Cangrelor had little effect on occlusion times, but addition of ADPstimulation to PRP sample inhibited occlusion essentially completely.

In the presence of cangrelor with ADP stimulation at the concentrationsshown to be inhibitory of platelets, thrombosomes (“thromb” in FIGS. 5-7) were not inhibited, indicating that thrombosomes can aid in a clotformation even in the presence of cangrelor at therapeutic levels.

The effect of cangrelor on thrombosomes under shear was evaluated byT-TAS®. FIG. 5 shows that thrombosomes (after 60, 90, or 115 minutes ofrehydration, as indicated) retain hemostatic function in the absence orpresence of cangrelor (1 uM), with ADP (10 uM) present. Unlikeplatelets, thrombosomes occlusion of the T-TAS® AR Chip is unaffected bythe antiplatelet effect of cangrelor+ADP. This suggests thrombosomeswill maintain expected function when infused into patients receivingcangrelor and similar agents. These results are further illustrated inFIGS. 6 and 7 .

In FIG. 6 , the AUC values (derived from the data in FIG. 5 ) areindicative of thrombus formation. There was no effect of cangrelor+ADPon thrombosome adhesion and occlusion of the T-TAS® AR Chip in plasma;thrombosomes caused a thrombus formation regardless of cangrelor andADP. The same dose of cangrelor and ADP completely inhibited freshlyharvested platelets.

In FIG. 7 , the time to occlusion (derived from the data in FIG. 5 ) ofthe thrombosomes on AR T-TAS® chip with drug treatment was evaluated.There was no effect from cangrelor+ADP on thrombosome time to occlusionusing the T-TAS® AR Chip in plasma. The same dose of cangrelor and ADPcompletely inhibited freshly harvested platelets.

Example 2. GPIIb-IIIa Inhibitors

The results that follow demonstrate the impact of thrombosomes in an invitro model of a patient taking a GPIIb-IIIa inhibitor. Eptifibatide, acommon antiplatelet drug, competitively inhibits the GPIIb-IIIa receptoron platelets which interact with fibrinogen and von Willebrand factor.

Eptifibatide is a peptide therapeutic that blocks the fibrin bindingrole of GPIIb-IIIa receptor on platelets. The drug is typicallyadministered via IV as a 180 μg/kg bolus followed by 2 μg/kg/mincontinuous infusion. The blood concentration of eptifibatide istypically about 1-2 μM. Bleeding time generally returns to normal withinabout 1 hour of drug stoppage.

Thrombosomes were prepared consistent with the procedure in Example 4.Transmission light aggregometry and T-TAS® experiments were carried outaccording to Example 4.

The aggregation of platelets (in platelet rich plasma) was evaluatedusing transmission light aggregometry. Eptifibatide completely inhibitedcollagen-induced (10 μg/mL) platelet aggregation in PRP at allconcentrations tested, as detected by light transmission aggregometry inPRP. (FIG. 8 ).

The effect of thrombosomes on shortening clotting times while in thepresence of eptifibatide was also studied. The ability of thrombosomesto recover occlusion times was studied on the T-TAS® system. The T-TAS®system measures occlusion time under shear forces with collagen andthromboplastin stimulation. The whole blood profile of occlusion and AUCon the AR T-TAS® chip lengthened and decreased, respectively, witheptifibatide. Eptifibatide extended the occlusion time of whole blood onthe T-TAS® AR Chip in a dose-dependent manner. In this experiment, wholeblood occluded at 8 minutes, and the occlusion time was extended to 16minutes with 6 μM eptifibatide (FIG. 9 ). Thrombosomes reversed theinhibitory effect of eptifibatide on thrombus formation. Eptifibatideinhibition of whole blood occlusion on the T-TAS® AR Chip was reversedby the addition of thrombosomes at approximately 200,000/4, (N=3). Whenthrombosomes (approximately 200 k/μL) were added to the sample of wholeblood inhibited with eptifibatide, the time to occlusion decreased to‘normal’ at 9 minutes (FIG. 10 ).

The area under the curve values with thrombosome treatment alsoincreased with thrombosomes compared to that of normal whole bloodsamples. FIG. 11 demonstrates the time to of occlusion of thethrombosomes on AR T-TAS® chip with drug treatment; eptifibatideinhibition of T-TAS® AR Chip occlusion was nearly entirely reversed bythe addition of thrombosomes (200,000/μL; N=3). In FIG. 12 , the areaunder the curve values were indicative of thrombus formation, wherethrombosomes returned inhibition by eptifibatide to normal levels;eptifibatide inhibition of platelet adhesion to and occlusion of theT-TAS® AR Chip is overcome by addition of thrombosomes (200,000/μL;N=3).

Thrombosomes, unlike platelets, are not inhibited in their ability toocclude under shear in the presence of eptifibatide (FIG. 13 ). FIG. 13shows profiles of thrombus formation of various lots of thrombosomes onAR T-TAS® system were unchanged with eptifibatide treatment.Thrombosomes in platelet poor plasma (PPP) were flowed through theT-TAS® AR Chip with and without 6 uM eptifibatide. There was no effectof eptifibatide on thrombosome adhesion and occlusion. All thrombosomeconcentrations were approximately 300,000/μL.

The AUC and occlusion values by T-TAS for thrombosomes (approximately300,000/μL) in plasma was the same with and without eptifibatide (FIG.14-15 ). FIG. 14 shows the area under the curve values were indicativeof thrombus formation, and no changes were observed with eptifibatide inplatelet-poor plasma. There was no effect of 6 uM eptifibatide on AUC ofthrombosomes T-TAS® AR Chip occlusion. FIG. 15 shows the time toocclusion of the thrombosomes on AR T-TAS® chip was unchanged witheptifibatide. There was no significant influence from 6 μM eptifibatideon thrombosomes occlusion time of the T-TAS® AR Chip in platelet-poorplasma.

Example 3. COX Inhibitors

The results that follow demonstrate the impact of thrombosomes in an invitro model of a patient taking a COX inhibitor. Aspirin, a commonantiplatelet drug, blocks the COX1 enzyme in platelets. COX1 isresponsible for converting arachidonic acid to prostaglandin.

Aspirin is an irreversible cyclooxygenase (COX) inhibitor. The COXenzyme in platelets is responsible for synthesis of thromboxane A2,prostaglandin E2, and prostacyclin (PGI2). Aspirin permanentlyinactivates the COX enzyme within platelets, and since platelets do nothave the nuclear material to synthesize new enzyme, new platelets mustbe produced to overcome the aspirin effect. Without thromboxane A2,prostaglandin E2 and prostacyclin (PGI2) platelets are limited in theirpro-aggregation activity. Many people are maintained on a low dose ofaspirin to prevent unwanted clotting events. Aspirin bioavailabilitylargely varies with administration route, with a single 500 mg dose IVat peaks of 500 μM and the same dose orally at 44 μM.

Thrombosomes were prepared consistent with the procedure in Example 4.Transmission light aggregometry and T-TAS® experiments were carried outaccording to Example 4.

Platelets will aggregate with collagen and arachidonic acid stimulation.

Stimulation by arachidonic acid can be completely inhibited whereascollagen stimulation aggregation can only be partially inhibited atconcentrations of 100-400 μM aspirin (FIG. 16 ). FIG. 16 shows lighttransmission aggregometry in PRP with collagen (10 ug/mL) andarachidonic acid (AA; 500 ug/mL), which induced platelet aggregation,and that aggregation was inhibited by all doses of aspirin (ASA) tested.Aspirin eliminated arachidonic acid induced platelet aggregationentirely. The PL chip system on the T-TAS® was used to emulate in vitroplatelet binding and aggregation due to the exposure of collagen in thevasculature under shear conditions. This action of platelets was largelylimited in the presence of 100 and 500 μM of aspirin but can be at leastpartially returned in the presence of thrombosomes (approximately200,000 to 400,000/μL; FIG. 17 ). FIG. 17 shows via area under the curvemeasurement of whole blood that thrombus formation on the PL T-TAS® chipwas inhibited by aspirin with partial return of thrombus formation withthrombosomes.

Example 4. Protocols

Generation of thrombosomes. Thrombosomes were prepared consistent withthe procedures described in U.S. Pat. No. 8,486,617 (such as, e.g.,Examples 1-5) and U.S. Pat. No. 8,097,403 (such as, e.g., Examples 1-3),incorporated herein by reference in their entirety.

Transmission Light Aggregometry

Plasma samples with platelet or thrombosomes or combination of both areloaded into cuvettes and placed into the aggregometry chambers. Thechambers warm the sample and provide constant stirring. The initiationof aggregation can be done by multiple types of inhibitor agents notlimited to thrombin, ADP, collagen and any agent known to stimulateplatelet aggregation. The samples can also have been taken as ex-vivo,or in-vitro supplemented with inhibitors. The instrument begins theassay by first recording the light transmission previous to stimulationfor 2 minutes. The stimulant of interest is then introduced by thetechnician and the change in light transmission is recorded over time.The increase in light transmission corresponds to increase in plateletaggregation.

Evaluation by T-TAS® using an AR chip. AR chips are characterized by asingle channel containing collagen and tissue factor; they can be usedto analyze clotting and platelet function.

The T-TAS® instrument was prepared for use according to themanufacturer's instructions. AR Chips (Diapharma Cat. #TC0101) and ARChip Calcium Corn Trypsin Inhibitor (CaCTI; Diapharma Cat. #TR0101) werewarmed to room temperature. 300 uL of rehydrated thrombosomes weretransferred to a 1.7 mL microcentrifuge tube and centrifuged at 3900g×10 minutes to pellet. The thrombosomes pellet was resuspended inGeorge King (GK) pooled normal human plasma or autologous plasma with orwithout autologous platelets to a concentration of approximately100,000-450,000/uL, as determined by AcT counts (Beckman Coulter AcTDiff 2 Cell Counter). 20 uL of CaCTI with 480 uL of thrombosomes samplein GK plasma were mixed with gentle pipetting. The sample was loaded andrun on the T-TAS® according to the manufacturer's instructions.

Evaluation by T-TAS® Using a PL Chip

PL chips are run similarly to AR chips, but this chip is only coatedwith collagen alone.

Thrombin Generation

Reagent Preparation. For thrombin generation, the following materialswere used from manufacturers, as follows: FluCa Kit (Diagnostica Stago,Cat. No. 86197), Thrombin calibrator (Diagnostica Stago, Cat. No.86197), PRP Reagent (Diagnostica Stago, Cat. No. 86196), OCTOPLAS®, asolvent detergent treated human pooled plasma (Octapharma, Cat. No.8-68209-952-04). All frozen reagents were thawed in a 37° C. water bathbefore use. All reagents were rehydrated with sterile water using thevolume printed on the reagent labels. Approximately 2 min afterrehydration, the reagents were mixed by inverting vials 5 times, so nochunks or powder left; vortexing was not used. This procedure wasrepeated approximately 10 minutes after rehydration. All reagents wereincubated at room temperature for another approximately 10 minutes(total of approximately 20 min after rehydration). A 30% solution ofOCTOPLAS® was prepared by mixing 4.66 ml of thrombosomes control buffer(Table B) with 2 ml of OCTOPLAS®.

TABLE B Thrombosomes Control Buffer Concentration (mg/mL, except whereComponent otherwise indicated) NaCl 6.08 KCl 0.28 HEPES 2.47 NaHCO₃ 0.77Dextrose 0.41 Trehalose 28.83 Ethanol 0.76% (v/v)

Sample Analysis—Plate preparation and testing. For experimentscontaining thrombosomes, a thrombosomes dilution series was generated(dilutions of 194.4K, 64.8K, 21.6K, and 7.2K per μL were typically used;cell counts are determined by flow cytometry) for each the experimentalthrombosomes and the reference thrombosomes. Thrombosomes wererehydrated unless indicated otherwise. The highest-concentrationdilution (e.g., 194.4 k thrombosomes) was prepared by combiningthrombosomes, OCTAPLAS®, and thrombosomes Control Buffer. The rest ofthe dilution series was prepared by serial 1:3 dilutions in OCTAPLAS®.For each test sample, 20 uL of PRP reagent was added to each sample well(of Immulon 2HB Clear, round-bottom 96-well plate (VWR, Cat. No.62402-954)) and 20 uL of Thrombin Calibrator was added to eachcalibrator well. To each sample well and calibrator well, 80 uL of eachof the thrombosomes dilution series was added. Continue until the lastdilution. The plate was then incubated in the Fluoroskan Ascent 96 wellfluorescent plate reader (Thrombinoscope) (ThermoFisher Scientific) for10 minutes. During this incubation phase, the FluCa solution wasprepared by adding 40 μL of FluCa substrate to the 1.6 ml of thawedFluo-Buffer, vortexing, and returning the solution to the water bath.When incubation was complete, the FluCa solution was added to theFluroskan instrument according to the manufacturer's instructions. Theplate fluorescence was monitored for 75 minutes at an interval of 20seconds and a temperature of 40-41° C.

Example 5

Additional experiments were carried out with cangrelor and aspirin.Thrombosomes were prepared consistent with the procedure in Example 4.Transmission light aggregometry, T-TAS®, and thrombin generationexperiments were carried out according to Example 4.

The effect of thrombosomes on the recovery of thrombus formation wasevaluated using T-TAS® technology and an AR chip. FIG. 18 shows theocclusion time of whole blood treated with various combinations ofthrombosomes (at a concentration of 250,000 thrombosomes per μL),aspirin (200 μM), cangrelor (1 μM), anti-Integrin alpha-2 (CD49B)antibody 6F1 (40 μg; seedshb.biology.uiowa.edu/integrin-alpha-2-alpha2beta1?sc=7&category=−107for product/manufacturer information), and anti-GPIIb/IIIa receptorantibody AP2 (20 ug/mL; seekerafast.com/product/2010/anti-glycoprotein-gpiiiagpiib-complex-ap-2-antibodyfor product/manufacturer information). FIG. 19 shows the occlusion overtime of untreated whole blood and whole blood treated with thrombosomes(at a concentration of 250,000 thrombosomes per a mixture containing 6F1(40 ug/mL; anti-CD49b), ASA (aspirin; 200 uM), and cangrelor (1 uM); ora combination thereof.

The effect of thrombosomes on the recovery of thrombus formation wasalso evaluated using T-TAS® technology and a PL chip. FIG. 20 shows theocclusion time of whole blood treated only with buffer, aspirin (500 oraspirin (500 μM) and thrombosomes (at a concentration of 250,000thrombosomes per μL). FIG. 21 shows the occlusion over time of wholeblood, whole blood treated with aspirin (500 μM), or aspirin (500 μM)and thrombosomes (250,000/μL). FIGS. 22 and 23 show similar experimentaldata using 100 μM aspirin instead of 500 μM aspirin.

The effect of aspirin treatment (concentration) on thrombin generationwas measured. Thrombosomes were evaluated at concentrations 1450, 1150,850, 650, 450, 150, 50, and 0 k/uL in PPP from patients taking babyaspirin daily and standard plasma (INR=1). FIG. 24 shows that the peakthrombin value of the aspirin plasma in absence of thrombosomes wasbelow the normal range (about 45 nM; normal range is about 66-166 nM),but with thrombosomes addition, it came back to being within the normalrange at even the lowest thrombosomes concentration used (50 k/μL). Thevalues again were saturated at about 800 k thrombosomes and went up to220 nM-5 times the value of this plasma in absence of thrombosomes(increase from 45 to 220 nM).

Example 6. Thrombosomes Reversed Prolonged PRP Occlusion Times Inducedby Cangrelor

Additional experiments were carried out with cangrelor. Thrombosomeswere prepared consistent with the procedure in Example 4. T-TAS® wascarried out according to Example 4.

FIGS. 25A and 25B show that platelet rich plasma treated with 100 ng/mLcangrelor and ADP extended occlusion times from 19 to 26 minutes on theT-TAS® flow system (collagen and tissue factor coated channel). Theaddition of 150 k/μL thrombosomes decreased the time back to 15.3minutes.

Example 7. Thrombosomes but not Random Donor Platelets (RDP) ReversedExtended Occlusion Times Induced by Tirofiban in PRP

Additional experiments were carried out with tirofiban. Thrombosomeswere prepared consistent with the procedure in Example 4. T-TAS® wascarried out according to Example 4. Random donor platelets were preparedfrom whole blood.

FIGS. 26A and 26B show that platelet rich plasma treated with 100 ng/mLtirofiban extended occlusion times from 18.43 to no occlusion on theT-TAS® flow system (collagen and tissue factor coated channel). Theaddition of 150 k/μL of thrombosomes decreased the time back to 12.94minutes but RDP only partially recovered at the same count.

Example 8. Thrombosomes but not Random Donor Platelets Reversed ExtendedOcclusion Times Induced by Eptifibatide in PRP

Additional experiments were carried out with eptifibatide. Thrombosomeswere prepared consistent with the procedure in Example 4. T-TAS® wascarried out according to Example 4. Random donor platelets were preparedfrom whole blood.

FIGS. 27A and 27B show that platelet rich plasma treated with 904eptifibatide extended occlusion times from 18.43 to over 30 minutes onthe T-TAS® flow system (collagen and tissue factor coated channel). Theaddition of 150 k/μL of thrombosomes decreased the time back to 11.56minutes but not occlusion seen with same number of RDP.

Example 9. Thrombosomes Reversed Extended Occlusion Times Induced by AP2(anti-GpIIb/IIIa) in PRP

Additional experiments were carried out with AP2. Thrombosomes wereprepared consistent with the procedure in Example 4. T-TAS® was carriedout according to Example 4. Random donor platelets were prepared fromwhole blood.

FIGS. 28A and 28B show that platelet rich plasma treated with 10 μg/mLAP-2 extended occlusion times from 18.43 to over 30 minutes on theT-TAS® flow system (collagen and tissue factor coated channel). Theaddition of 150 k/μL of thrombosomes decreased the time back to 13.14minutes and occlusion was seen at 17.43 minutes same number of RDP.

Example 10. Thrombosomes Reversed Prolonged Occlusion in PRP fromSubjects on Aspirin Therapy

Additional experiments were carried out with aspirin. Thrombosomes wereprepared consistent with the procedure in Example 4. T-TAS® was carriedout according to Example 4. Random donor platelets were prepared fromwhole blood. The subject was on a standard dose of 81 mg/day of aspirin.

FIGS. 29A and 29B show that platelet rich plasma taken from an aspirinpatient failed to occlude on the T-TAS® flow system (collagen and tissuefactor coated channel). The addition of 200 k/μL of thrombosomesreturned to normal occlusion time to 16 minutes.

Example 11. Thrombosomes Restore Thrombin Generation in Ex-Vivo AspirinPlatelet Rich Plasma

Additional experiments were carried out with aspirin. Thrombosomes wereprepared consistent with the procedure in Example 4. Thrombin generationwas carried out according to Example 4.

FIG. 30A shows Thrombin generation of platelet rich plasma from aspirinpatient verses normal stimulated with PRP reagent was reversed with 50k/μL of thrombosomes. FIG. 30B shows the change from and return tonormal thrombin production, time to peak production, and lag time inthree repeat aspirin ex-vivo samplings with thrombosomes (50 k/μL). (n=3thrombosome lots, n=2 individuals).

Example 12. Thrombosomes Restore Hemostasis in PRP from Subject on NSAIDIbuprofen Therapy

Additional experiments were carried out with ibuprofen, an NSAID.

Thrombosomes were prepared consistent with the procedure in Example 4.Aggregometry and T-TAS® were carried out according to Example 4.

Platelet rich plasma was taken from subject on 800 mg ibuprofen. FIG.31A shows that a lack of aggregation in response to arachidonic acidconfirms NSAID presence in the PRP. FIG. 31B shows occlusion on theT-TAS® flow system (collagen and tissue factor coated channel); PRP fromthe ibuprofen patient demonstrated occlusion, while addition of ADPabolished occlusion. The addition of 150 k/μL thrombosomes restoredocclusion.

Example 13. Thrombosomes® Restore Bleeding Time in NOD-SCID Mice Treatedwith Supra-Pharmacologic Clopidogrel

Additional experiments were carried out with clopidogrel. Thrombosomeswere prepared consistent with the procedure in Example 4.

The mouse was treated with clopidogrel for 5 days. The mouse wasanesthetized, the tail end was snipped off followed by thrombosomesbeing immediately administered. The time from tail snip to tail stopbleeding was recorded by visual inspection.

NOD/SCID mice were treated with ˜3 times the clinical dose ofclopidogrel for 5 days then assessed in the tail-snip bleed model. Thebleed time (min) was extended to 17.8 minutes with clopidogrel treatmentverses untreated at 9 minutes (data not shown). Treatment with 8 μL/gramof thrombosomes (1.8×10{circumflex over ( )}9 particles/mL at 200 μL)decreased bleeding to 12.31 minutes (FIG. 32 ).

Although the foregoing description is directed to the preferredembodiments of the invention, it is noted that other variations andmodifications will be apparent to those skilled in the art, and may bemade without departing from the spirit or scope of the invention.Moreover, features described in connection with one embodiment of theinvention may be used in conjunction with other embodiments, even if notexplicitly stated above. Furthermore, one having ordinary skill in theart will readily understand that the invention as discussed above may bepracticed with steps in a different order, and/or with hardware elementsin configurations which are different than those which are disclosed.Therefore, although the invention has been described based upon thesepreferred embodiments, it would be apparent to those of skill in the artthat certain modifications, variations, and alternative constructionswould be apparent, while remaining within the spirit and scope of theinvention. Embodiments of the invention so claimed are inherently orexpressly described and enabled herein. In order to determine the metesand bounds of the invention, therefore, reference should be made to theappended claims.

What is claimed is:
 1. A method of restoring normal hemostasis in asubject, the method comprising administering to the subject in needthereof an effective amount of a composition comprising platelets orplatelet derivatives and an incubating agent comprising one or moresalts, a buffer, optionally a cryoprotectant, and optionally an organicsolvent, wherein the subject has been treated or is being treated withan antiplatelet agent.
 2. A method of restoring normal hemostasis in asubject, the method comprising administering to the subject in needthereof an effective amount of a composition prepared by a processcomprising incubating platelets with an incubating agent comprising oneor more salts, a buffer, optionally a cryoprotectant, and optionally anorganic solvent, to form the composition, wherein the subject has beentreated or is being treated with an antiplatelet agent.
 3. A method oftreating a coagulopathy in a subject, the method comprisingadministering to the subject in need thereof an effective amount of acomposition comprising platelets or platelet derivatives and anincubating agent comprising one or more salts, a buffer, optionally acryoprotectant, and optionally an organic solvent, wherein the subjecthas been treated or is being treated with an antiplatelet agent.
 4. Amethod of treating a coagulopathy in a subject, the method comprisingadministering to the subject in need thereof an effective amount of acomposition prepared by a process comprising incubating platelets withan incubating agent comprising one or more salts, a buffer, optionally acryoprotectant, and optionally an organic solvent, to form thecomposition, wherein the subject has been treated or is being treatedwith an antiplatelet agent.
 5. A method of preparing a subject forsurgery, the method comprising administering to the subject in needthereof an effective amount of a composition comprising platelets orplatelet derivatives and an incubating agent comprising one or moresalts, a buffer, optionally a cryoprotectant, and optionally an organicsolvent, wherein the subject has been treated or is being treated withan antiplatelet agent.
 6. A method of preparing a subject for surgery,the method comprising administering to the subject in need thereof aneffective amount of a composition prepared by a process comprisingincubating platelets with an incubating agent comprising one or moresalts, a buffer, optionally a cryoprotectant, and optionally an organicsolvent, to form the composition, wherein the subject has been treatedor is being treated with an antiplatelet agent.
 7. The method of any oneof claims 5-6, wherein the surgery is an emergency surgery.
 8. Themethod of any one of claims 5-6, wherein the surgery is a scheduledsurgery.
 9. The method of any one of claims 1-8, wherein the subject isbeing treated with an antiplatelet agent.
 10. The method of claim 9,wherein treatment with the antiplatelet agent is stopped.
 11. The methodof claim 9, wherein treatment with the antiplatelet agent is continued.12. A method of ameliorating the effects of an antiplatelet agent in asubject, the method comprising administering to the subject in needthereof an effective amount of a composition comprising platelets orplatelet derivatives and an incubating agent comprising one or moresalts, a buffer, optionally a cryoprotectant, and optionally an organicsolvent.
 13. A method of ameliorating the effects of an antiplateletagent in a subject, the method comprising administering to the subjectin need thereof an effective amount of a composition prepared by aprocess comprising incubating platelets with an incubating agentcomprising one or more salts, a buffer, optionally a cryoprotectant, andoptionally an organic solvent, to form the composition.
 14. The methodof claim 12 or claim 13, wherein the effects of the antiplatelet agentare the result of an overdose of the antiplatelet agent.
 15. The methodof any one of claims 1-14, wherein the composition further comprises ananti-fibrinolytic agent.
 16. The method of claim 15, wherein theanti-fibrinolytic agent is selected from the group consisting ofε-aminocaproic acid (EACA), tranexamic acid, aprotinin,aminomethylbenzoic acid, fibrinogen, and a combination thereof.
 17. Themethod of claim 15 or claim 16, wherein the platelets or plateletderivatives are loaded with the anti-fibrinolytic agent.
 18. The methodof any one of claims 1-17, wherein the antiplatelet agent is selectedfrom the group consisting of aspirin, cangrelor, ticagrelor,clopidogrel, prasugrel, eptifibatide, tirofiban, abciximab, asupplement, and a combination thereof.
 19. The method of any one ofclaims 1-17, wherein the antiplatelet agent is selected from the groupconsisting of aspirin, cangrelor, ticagrelor, clopidogrel, prasugrel,eptifibatide, tirofiban, abciximab, terutroban, picotamide, elinogrel,ticlopidine, ibuprofen, vorapaxar, atopaxar, and a combination thereof.20. The method of any one of claims 1-17, wherein the antiplatelet agentis selected from the group consisting of aspirin, cangrelor, ticagrelor,clopidogrel, prasugrel, eptifibatide, tirofiban, abciximab, terutroban,picotamide, elinogrel, ticlopidine, ibuprofen, vorapaxar, atopaxar,cilostazol, prostaglandin E1, epoprostenol, dipyridamole, treprostinilsodium, sarpogrelate, and a combination thereof.
 21. The method of anyone of claims 1-20, wherein administering comprises administeringtopically.
 22. The method of any one of claims 1-20, whereinadministering comprises administering parenterally.
 23. The method ofany one of claims 1-20, wherein administering comprises administeringintravenously.
 24. The method of any one of claims 1-20, whereinadministering comprises administering intramuscularly.
 25. The method ofany one of claims 1-20, wherein administering comprises administeringintrathecally.
 26. The method of any one of claims 1-20, whereinadministering comprises administering subcutaneously.
 27. The method ofany one of claims 1-20, wherein administering comprises administeringintraperitoneally.
 28. The method of any one of claims 1-27, wherein thecomposition is dried prior to the administration step.
 29. The method ofclaim 28, wherein the composition is rehydrated following the dryingstep.
 30. The method of any one of claims 1-28, wherein the compositionis freeze-dried prior to the administration step.
 31. The method ofclaim 30, wherein the composition is rehydrated following thefreeze-drying step.
 32. The method of any one of claims 1-31, whereinthe incubating agent comprises one or more salts selected from phosphatesalts, sodium salts, potassium salts, calcium salts, magnesium salts,and a combination of two or more thereof.
 33. The method of any one ofclaims 1-32, wherein the incubating agent comprises a carrier protein.34. The method of any one of claims 1-33, wherein the buffer comprisesHEPES, sodium bicarbonate (NaHCO₃), or a combination thereof.
 35. Themethod of any one of claims 1-34, wherein the composition comprises oneor more saccharides.
 36. The method of claim 35, wherein the one or moresaccharides comprise trehalose.
 37. The method of claim 35 or claim 36,wherein the one or more saccharides comprise polysucrose.
 38. The methodof any one of claims 35-37, wherein the one or more saccharides comprisedextrose.
 39. The method of any one of claims 1-38, wherein thecomposition comprises an organic solvent.
 40. The method of any ofclaims 1-39, wherein the platelets or platelet derivatives comprisethrombosomes.
 41. The method of any one of claim 1-6, 12, or 13, whereinthe effective amount of the composition comprises 1.0×10⁷ particles toabout 1.0×10¹⁰ particles of platelet derivatives/kg of the subject.